Pd-l1 and pd-l2-based fusion proteins and uses thereof

ABSTRACT

Provided are fusion proteins comprising a first domain and a second domain, wherein the first domain comprises a polypeptide that binds to and triggers PD-1 and the second domain comprises a polypeptide that binds to and triggers a TRAIL receptor or Fas. In some embodiments, the polypeptide that binds to and triggers PD-1 comprises at least a portion of the extracellular domain of PD-L1 or PD-L2 and the second domain comprises at least a portion of the extracellular domain of TRAIL or Fas ligand. Also provided are methods for treating autoimmune, alloimmune or inflammatory diseases, and methods for treating cancer, using the fusion proteins.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.61/759,287, filed Jan. 31, 2013, the entire disclosure of which isincorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 30, 2014, isnamed 37075_(—)0291_(—)00_WO_SeqListing_ST25, and is 64,174 bytes insize.

FIELD OF THE INVENTION

The present invention relates to fusion proteins comprising a firstdomain and a second domain, wherein the first domain is a polypeptidethat binds to and triggers PD-1, such as PD-L1 or PD-L2, and the seconddomain is a polypeptide that binds to and triggers a TRAIL receptor orFas, such as TRAIL or Fas ligand.

BACKGROUND OF THE INVENTION

A complex interplay of positive and negative signals regulates T cellactivation and maintenance of T cell effector function. Members of theTNF ligand/TNF receptor superfamily figure prominently in this matrix ofsignals, bridging cells of the immune system, as well as with cells ofother organ systems. In so doing, TNF superfamily members contribute toboth tissue homeostasis and pathogenesis, via effects on cell survivaland death, cellular differentiation, and inflammation. From thestandpoint of autoimmune pathogenesis, an interesting member of the TNFligand superfamily is TNF-related apoptosis-inducing ligand (TRAIL).

TRAIL binds to a number of different cognate receptors of the TNFreceptor superfamily, some leading to triggering of intracellularsignaling pathways and others simply acting as decoy receptors. Thetriggering receptors in humans are TRAIL-R1, TRAIL-R2, andosteoprotegrin, and in mice the sole triggering receptor is DR5.Virtually all cells of the immune system (T lymphocytes, B lymphocytes,natural killer cells, dendritic cells, monocytes, granulocytes)upregulate surface TRAIL and/or release soluble TRAIL stored insecretory vesicles in response to interferon and other activationsignals. TRAIL inhibits autoimmunity in several animal models. Evidencefor TRAIL's capacity to inhibit experimental autoimmune encephalitis(EAE), a murine model for multiple sclerosis (MS), has come fromexperiments invoking TRAIL−/−knockout mice, soluble TRAIL receptor(sDR5) or neutralizing anti-TRAIL mAb capable of blocking TRAILfunction, and embryonic stem cell-derived dendritic cells co-expressingTRAIL and pathogenic MOG (myelin oligo-dendrocyte glycoprotein peptide).Interestingly, in MS patients, soluble TRAIL has emerged as a responsemarker for IFN-β therapy, with those most likely to respond to treatmentshowing early and sustained soluble TRAIL induction after therapy. Yet,TRAIL's impact on MS/EAE may be more complex, for example, thesuggestion that TRAIL may promote brain cell apoptosis. Both TRAIL andFasL have been implicated in inhibition of T cells and the induction ofapoptosis in T cells.

Apoptosis or programmed cell death (PCD) is a form of cell death whichis essential for the regulation of cellular homeostasis. In the immunesystem, Fas (CD95) receptor and its ligand, FasL (CD95L), participate invarious processes involved in the induction of apoptosis, includingimmune cell-mediated cytotoxicity, and in the regulation of cellularimmune responses. FasL is a member of the tumor necrosis factorsuperfamily and is expressed by a restricted subset of immune cells,including monocytes, NK cells, and activated B and T cells. On the cellsurface, FasL is oriented as a type II membrane protein with trimericcomplexes. Metalloproteinase cleavage of membrane-associated FasLreleases soluble FasL (sFasL) trimmers from the membrane. The FasLmolecule triggers Fas-dependent PCD.

One of the newer pathways that provide costimulatory and inhibitorysecond signals to T cells is represented by the programmed death 1(PD-1; also known as CD279) receptor and its ligands, PD-L1 (B7-H1;CD274) and PD-L2 (B7-DC; CD273). PD-1 is a member of the CD28/CTL4family that is expressed on activated, but not resting T cells(Nishimura et al. (1996) Int. Immunol. 8:773). Ligation of PD-1 by itsligands mediates an inhibitory signal that results in reduced cytokineproduction, and reduced T cell survival (Nishimura et al. (1999)Immunity 11:141; Nishimura et al. (2001) Science 291:319, Chemitz et al.(2004) J. Immunol. 173:945).

PD-L1 is a B7 family member that is expressed on many cell types,including antigen presenting cells (APCs) and activated T cells(Yamazaki et al. (2002) J. Immunol. 169:5538). PD-L1 binds to both PD-1and B7-1. Both binding of T-cell expressed B7-1 by PD-L1 and binding ofT-cell-expressed PD-L1 by B7 result in T cell inhibition (Butte et al.(2007) Immunity 27:111). There is also evidence that, like other B7family members, PD-L1 can also provide costimulatory signals to T cells(Subudhi et al. (2004) J. Clin. Invest. 113:694; Tamura et al. (2001)Blood 97:1809).

PD-L2 is a B7 family member expressed on various APCs, includingdendritic cells, macrophages and bone-marrow derived mast cells (Zhonget al. (2007) Eur. J. Immunol. 37:2405). APC-expressed PD-L2 is able toboth inhibit T cell activation through ligation of PD-1 and costimulateT cell activation, through a PD-1 independent mechanism (Shin et al.(2005) J. Exp. Med. 201:1531). In addition, ligation of dendriticcell-expressed PD-L2 results in enhanced dendritic cell cytokineexpression and survival (Radhakrishnan et al. (2003) J. Immunol.37:1827; Nguyen et al. (2002) J. Exp. Med. 196:1393). The structure andexpression of PD-1, PD-L1 and PD-L2, as well as signalingcharacteristics and functions of these molecules in the context ofregulating T cell activation and tolerance (e.g., therapeutic effects)are reviewed in greater detail in Kier et al. (2008) Ann. Rev. Immunol.26:677, which is herein incorporated by reference in its entirety.

SUMMARY OF THE INVENTION

Provided is a fusion protein comprising a first domain and a seconddomain, wherein the first domain comprises a polypeptide that binds toand triggers PD-1 and the second domain comprises a polypeptide thatbinds to and triggers a TRAIL receptor or Fas. In some embodiments, thepolypeptide that binds to and triggers PD-1 comprises at least a portionof the extracellular domain of PD-L1 or PD-L2 and the second domaincomprises at least a portion of the extracellular domain of TRAIL or Fasligand. In further embodiments, the fusion protein comprises at least aportion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11 or SEQ ID NO:12.

In certain embodiments, provided is a fusion protein according to anyone of the preceding embodiments, comprising a first domain and a seconddomain, wherein the first domain comprises a portion of theextracellular domain of PD-L1 or PD-L2 comprising at least 20 contiguousamino acids of said extracellular domain, and the second domaincomprises a portion of the extracellular domain of TRAIL or Fas ligandcomprising at least 20 contiguous amino acids of said extracellulardomain, and the first and second domain are connected via an optionallinker. In some embodiments, the first domain comprises human PD-L1 orhuman PD-L2, or a fragment thereof capable of binding to and triggeringPD-1, and the second domain comprises human TRAIL or human Fas ligand,or a fragment thereof. In further embodiments, the first domain and thesecond domain are connected via a linker. In yet further embodiments,the linker comprises a protein linker.

Further provided is the fusion protein of any one of the precedingembodiments, in a pharmaceutically acceptable carrier.

Also provided is a pharmaceutical composition comprising the fusionprotein of any one of the preceding embodiments.

Further provided is a method of treating an autoimmune, alloimmune orinflammatory disease in a patient comprising administering the fusionprotein of any one of the preceding embodiments to a patient in need ofsuch treatment. In some embodiments, the autoimmune disease is multiplesclerosis. In further embodiments of the method, the administration isparenteral.

Also provided is a method of inhibiting proliferation anddifferentiation of T cells, B cells, mast cells, antigen presentingcells, dendritic cells or NK cells in a patient, the method comprisingthe step of administering the fusion protein of any one of the precedingembodiments to a patient in need of such treatment.

Also provided is a method of treating cancer in a patient comprisingadministering the fusion protein of any one of the preceding embodimentsto a patient in need of such treatment.

Also provided is a method of treating autoimmune disease, alloimmunedisease, inflammatory disease or cancer in a patient by administering tosaid patient an effective amount of a genetic sequence encoding thefusion protein of any one of the preceding embodiments.

Further provided is a fusion protein of any one of the precedingembodiments, for use in (i) medicine, (ii) treating an autoimmune,alloimmune or inflammatory, (iii) inhibiting proliferation anddifferentiation of T cells, B cells, mast cells, antigen presentingcells, dendritic cells or NK cells, or (iii) treating cancer. Alsoprovided is a genetic sequence encoding a fusion protein of any one ofthe preceding embodiments, for use in medicine, or for use in treatingautoimmune disease, alloimmune disease, inflammatory disease or cancer.

DEFINITIONS

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise.

The term “about” will be understood by persons of ordinary skill in theart and will vary to some extent depending on the context in which it isused. As used herein, “about” is meant to encompass variations of ±20%or ±10%, more preferably ±5%, even more preferably ±1%, and still morepreferably ±0.1%.

As used herein, the term “fusion protein” or fusion polypeptide is apolypeptide comprised of at least two polypeptides and optionally alinking sequence, and that are operatively linked into one continuousprotein. The two polypeptides linked in a fusion protein are typicallyderived from two independent sources, and therefore a fusion proteincomprises two linked polypeptides not normally found linked in nature.Typically, the two polypeptides can be operably attached directly by apeptide bond.

As used herein, “trigger” with respect to a receptor, such as TRAILreceptor or Fas, refers to the biological change that occurs uponligation of the receptor by an agonist ligand. Biological changes thatcan occur when a receptor is triggered include, but are not limited to,one or more of: receptor interaction with one or more intracellularadaptors and effector molecules; induction of a signaling cascade;modified expression of molecules; release of cytokines and/orchemokines; activation of caspases; activation of transcription factors;changes in protein modification such as a phosphorylation; activation ofsignal transduction pathways such as NF-κB and P13K; induction ofdownstream effects on transcriptional, translational, andpost-translational control mechanisms affecting one or more genes and/orproteins expressed by the cell.

As used herein, the term “immune disease” and like terms means adisease, disorder or condition caused by the immune system of an animal,including autoimmune disorders. Immune disorders include those diseases,disorders or conditions that have an immune component and those that aresubstantially or entirely immune system-mediated.

As used herein, the term “alloimmune disease” refers to when there is ahost immune response to foreign antigens of another individual (forexample, major or minor histocompatibility alloantigens), for examplewhen there is a host-versus-graft rejection, or alternatively when thereis graft-versus-host disease, wherein engrafted immune cells mediatedeleterious effects against the host receiving the graft.

As used herein, the term “autoimmune disease” refers to a disease,disorder or condition that results from an aberrant immune response thatresults from the failure of an organism in recognizing its ownconstituent parts as self, which allows an immune response against itsown cells and tissues.

As used herein, an “inflammatory disease” means a disease, disorder orcondition characterized by inflammation of body tissue or having aninflammatory component., typically due to infiltration of tissue byimmune cells. These disorders include both local inflammatory responsesand systemic inflammation.

The term “operably linked,” as used herein, indicates that two molecules(e.g., polypeptides) are attached so as to each retain biologicalactivity. Two molecules are “operably linked” whether they are attacheddirectly or indirectly (e.g., via a linker).

The term “linker,” as used herein, refers to a peptide that isoptionally located between two amino acid sequences in the fusionprotein of the invention.

As used herein, “biologically active” or “immunologically active” asapplied to fusion proteins refers to fusion proteins according to thepresent invention having a similar structural function (but notnecessarily to the same degree), and/or similar regulatory function (butnot necessarily to the same degree), and/or similar biochemical function(but not necessarily to the same degree) and/or immunological activity(but not necessarily to the same degree) as the individual wild typeproteins which are the building blocks of the fusion proteins of thepresent invention.

As used herein, a “deletion” in an amino acid sequence or polypeptide isdefined as a change in amino acid sequence in which one or more aminoacid residues are absent as compared to the wild-type protein.

As used herein an “insertion” or “addition” in an amino acid sequence orpolypeptide is a change in an amino acid sequence that has resulted inthe addition of one or more amino acid residues as compared to thewild-type protein.

As used herein “substitution” in an amino acid sequence or polypeptideresults from the replacement of one or more amino acids by differentamino acids, respectively, as compared to the wild-type protein.

As used herein, a “trimerization domain” refers to an amino acidsequence within a polypeptide that promotes assembly of the polypeptideinto trimers. For example, a trimerization domain can promote assemblyof a protein into trimers via associations with other trimerizationdomains (of additional polypeptides with the same or a different aminoacid sequence). The term is also used to refer to a polynucleotide thatencodes such a peptide or polypeptide.

As used herein, the term “variant” with respect to an amino acidsequence or polypeptide means any polypeptide having a substitution of,deletion of or addition of one (or more) amino acid from or to thesequence, including allelic variations, as compared with the wild-typeprotein, so long as the resultant variant fusion protein retains atleast 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of thebiological or immunologic activity as compared to the wild-type proteinsas used in the present invention. Additionally, while in general it isdesirable for variants to show enhanced ability for binding to a givenmolecule, in some embodiments variants may be designed with slightlyreduced activity as compared to other fusion proteins of the invention,for example, in instances in which one would purposefully want toattenuate activity, for example, to diminish neurotoxicity. Moreover,variants or derivatives can be generated that would bind moreselectively to one of the TRAIL receptor variants (there are four TRAILreceptors in humans). Furthermore, variants or derivatives can begenerated that would have altered multimerization properties. Whenengineering variants, this could be done for either the entire TRAILextracellular domain, or for that component of the extracellular domainthat is incorporated within the fusion protein itself.

In one embodiment, variants of the fusion proteins of the presentinvention will have at least 80% or greater sequence identity orhomology, as those terms are understood in the art, to SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ IDNO:12, more preferably at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or even 99% sequence identity to SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQID NO:12.

Sequence identity or homology can be determined using standardtechniques known in the art, such as the Best Fit sequence programdescribed by Devereux et al., Nucl. Acid Res. 12:387-395 (1984) or theBLASTX program (Altschul et al., J. Mol. Biol. 215, 403-410). Thealignment may include the introduction of gaps in the sequences to bealigned. In addition, for sequences which contain either more or feweramino acids than the proteins disclosed herein, it is understood thatthe percentage of homology will be determined based on the number ofhomologous amino acids in relation to the total number of amino acids.

Additionally, while in general it is desirable for variants to showenhanced ability for binding to a given molecule, in some embodimentsvariants may be designed with slightly reduced activity as compared toother fusion proteins of the invention, for example, in instances inwhich one would purposefully want to attenuate activity, for example, todiminish neurotoxicity. Moreover, variants or derivatives can begenerated that would bind more selectively to one of the TRAIL receptorvariants (there are two TRAIL receptors in humans that induceapoptosis). Furthermore, variants or derivatives can be generated thatwould have altered multimerization properties. When engineeringvariants, this could be done for either the entire TRAIL extracellulardomain, or for that component of the extracellular domain that isincorporated within the fusion protein itself.

Preferably, variants or derivatives of the fusion proteins of thepresent invention maintain the hydrophobicity/hydrophilicity of theamino acid sequence. Conservative amino acid substitutions may be made,for example from 1, 2 or 3 to 10, or 30 substitutions provided that themodified sequence retains the ability to act as a fusion protein inaccordance with present invention. Amino acid substitutions may includethe use of non-naturally occurring analogues, for example to increaseblood plasma half-life.

Conservative substitutions are known in the art, for example accordingto the table below. Amino acids in the same block in the second columnand preferably in the same line in the third column may be substitutedfor each other:

ALIPHATIC Non-polar GAPILV Polar- CSTM Uncharged NQ Polar-charged DE KRAROMATIC HFWY

The term “derivative” as used herein in relation to the amino acidsequence means chemical modification of a fusion protein of theinvention.

Non-limiting examples of such modifications may include but are notlimited to aliphatic esters or amides of the carboxyl terminus or ofresidues containing carboxyl side chains, O-acyl derivatives of hydroxylgroup-containing residues, and N-acyl derivatives of the amino-terminalamino acid or amino-group containing residues, e.g., lysine or arginine.

Additional modifications can include, for example, production of afusion protein conjugated with polyethylene glycol (PEG), or addition ofPEG during chemical synthesis of a polypeptide of the invention.

Modifications of polypeptides or portions thereof can also includereduction/alkylation; chemical coupling to an appropriate carrier ormild formalin treatment.

Other derivatives of the fusion proteins of the present inventioninclude incorporation of unnatural amino acid residues, orphosphorylated amino acid residues such as phosphotyrosine,phosphoserine or phosphothreonine residues. Other potentialmodifications include sulfonation, biotinylation, or the addition ofother moieties, particularly those which have molecular shapes similarto phosphate groups.

Derivatives also include polypeptides modified by glycosylation. Thesecan be made by modifying glycosylation patterns during synthesis andprocessing in various alternative eukaryotic host expression systems, orduring further processing steps. Methods for producing glycosylationmodifications include exposing the fusion proteins to glycosylatingenzymes derived from cells that normally carry out such processing, suchas mammalian glycosylation enzymes. Alternatively, deglycosylationenzymes can be used to remove carbohydrates attached during productionin eukaryotic expression systems. Additionally, one can also modify thecoding sequence so that glycosylations site(s) are added orglycosylation sites are deleted or disabled. Furthermore, if noglycosylation is desired, the proteins can be produced in a prokaryotichost expression system.

Variants and/or derivatives of the fusion proteins of the invention canbe prepared by chemical synthesis or by using site-directed mutagenesis(Gillman et al., Gene 8:81 (1979); Roberts et at, Nature 328:731 (1987)or Innis (Ed.), 1990, PCR Protocols: A Guide to Methods andApplications, Academic Press, New York, N.Y.) or the polymerase chainreaction method (PCR; Saiki et al, Science 239:487 (1988)), asexemplified by Daugherty et at (Nucleic Acids Res. 19:2471 (1991)) tomodify nucleic acids encoding the complete receptors.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

The term “expression vector” as used herein refers to a vectorcontaining a nucleic acid sequence coding for at least part of a geneproduct capable of being transcribed. In some cases, RNA molecules arethen translated into a protein, polypeptide, or peptide. Expressionvectors can contain a variety of control sequences, which refer tonucleic acid sequences necessary for the transcription and possiblytranslation of an operatively linked coding sequence in a particularhost organism. In addition to control sequences that governtranscription and translation, vectors and expression vectors maycontain nucleic acid sequences that serve other functions as well.

An “isolated nucleic acid” refers to a nucleic acid segment or fragmentwhich has been separated from sequences which flank it in a naturallyoccurring state, i.e., a DNA fragment which has been removed from thesequences which are normally adjacent to the fragment, i.e., thesequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, i.e., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (i.e.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used, “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means.

The term “polypeptide” as used herein is defined as a chain of aminoacid residues, usually having a defined sequence. As used herein theterm polypeptide is mutually inclusive of the terms “peptide” and“protein”.

As used herein, a polypeptide is “soluble” when it lacks anytransmembrane domain or peptide domain that anchors or integrates thepolypeptide into the membrane of a cell expressing such polypeptide. Inparticular, the soluble proteins useful as components in the fusionprotein of the invention may exclude transmembrane and intracellulardomains. The soluble proteins may comprise substantially all of anectodomain or may comprise a fragment thereof possessing the requiredagonist function, e.g., a functional fragment.

The term “promoter” as used herein is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa polynucleotide sequence.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell under most or allphysiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell substantially only whenan inducer which corresponds to the promoter is present in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a cell substantiallyonly if the cell is a cell of the tissue type corresponding to thepromoter.

The term “RNA” as used herein is defined as ribonucleic acid.

The term “recombinant DNA” as used herein is defined as DNA produced byjoining pieces of DNA from different sources.

The term “recombinant polypeptide” as used herein is defined as apolypeptide produced by using recombinant DNA methods.

As used herein, a “therapeutically effective amount” is the amount of acomposition sufficient to provide a beneficial effect to a mammal towhich the composition is administered. An therapeutically effectiveamount of a fusion protein of the invention is an amount that willameliorate one or more of the well known parameters that characterizemedical conditions caused by autoimmune disease, alloimmune disease,inflammatory disease or cancer. Many such parameters and conditions havebeen described and are well known to the skilled artisan. Atherapeutically effective amount, in the context of a cancer, forexample, will be the amount of fusion protein that is sufficient toaccomplish one or more of the following: decrease the severity ofsymptoms; decrease tumor size; decrease rate of tumor growth; decreasethe duration of disease exacerbations; increase the frequency andduration of disease remission/symptom-free periods; prevent fixedimpairment and disability; and/or prevent/attenuate chronic progressionof the disease.

“Treating” or “treatment” refers to therapeutic treatment, wherein theobject is to prevent or slow down (lessen) the targeted pathologiccondition or disorder. A subject is “treated” if: after receiving atherapeutic amount of a fusion protein of the invention according to themethods of the present invention, the subject shows observable and/ormeasurable reduction in or absence of one or more signs and symptoms ofthe particular disease. For example, for cancer, reduction in the numberof cancer cells or absence of the cancer cells; reduction in the tumorsize; inhibition (i.e., slow to some extent and preferably stop) oftumor metastasis; inhibition, to some extent, of tumor growth; increasein length of remission, and/or relief to some extent, one or more of thesymptoms associated with the specific cancer; reduced morbidity andmortality, and improvement in quality of life issues. Treatment canachieve a complete response, defined as disappearance of all signs ofcancer, or a partial response, wherein the size of the tumor isdecreased, preferably by more than 50%, more preferably by 75%. Apatient is also considered treated if the patient experiences astabilization of disease.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

The phrase “under transcriptional control” or “operatively linked” asused herein with respect to polynucleotides means that the promoter isin the correct location and orientation in relation to a polynucleotideto control the initiation of transcription by RNA polymerase andexpression of the polynucleotide.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

The term “TRAIL receptor” as used herein refers to a receptor that bindsto TRAIL ligand and induces or triggers apoptosis. In some embodiments,the TRAIL receptor is DR4 (TRAILR1). In some embodiments, the TRAILreceptor is DR5 (TRAILR2). The term “TRAIL receptor” as used herein doesnot refer to the receptors DcR1 (TRAILR3) and DcR2. DcR1 does notcontain a cytoplasmic domain, and DcR2 (TRAILR4) contains a truncateddeath domain. DcR1 functions as a TRAIL-neutralizing decoy-receptor. Thecytoplasmic domain of DcR2 is functional and activates NFkappaB. Incells expressing DcR2, TRAIL binding therefore activates NFkappaB,leading to transcription of genes known to antagonize the deathsignaling pathway and/or to promote inflammation.

The term “Fas” or “Fas receptor” as used herein refers to a receptorthat binds to Fas ligand (FasL) and induces or triggers apoptosis.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “about”, even if the term does notexpressly appear.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

Where any amino acid sequence is specifically referred to by a SwissProt. or GENBANK Accession number, the sequence is incorporated hereinby reference. Information associated with the accession number, such asidentification of signal peptide, extracellular domain, transmembranedomain, promoter sequence and translation start, is also incorporatedherein in its entirety by reference.

As envisioned in the present invention with respect to the disclosedcompositions of matter and methods, in one aspect the embodiments of theinvention comprise the components and/or steps disclosed therein. Inanother aspect, the embodiments of the invention consist essentially ofthe components and/or steps disclosed therein. In yet another aspect,the embodiments of the invention consist of the components and/or stepsdisclosed therein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, in one aspect, a fusion protein thatacts on the PD-1 and TRAIL or Fas signaling axis, for example a fusionprotein comprising a first domain that comprises PD-L1 or PD-L2; and asecond domain that comprises a polypeptide that binds to and triggers aTRAIL receptor or a Fas receptor. In particular, the first domaincomprises at least a portion of the extracellular domain of PD-L1 orPD-L2, and the second domain comprises at least a portion of apolypeptide that can bind to TRAIL receptor or Fas receptor and directinhibitory signals through cognate receptors on T cells or other cellsbearing a TRAIL receptor or a Fas receptor.

The first domain and the second domain need be in no particular order.In some preferred embodiments, the first domain is at the N-terminus ofthe protein, and the second domain is at the C-terminus of the protein.In some embodiments, the second domain is at the N-terminus of theprotein, and the second domain is at the C-terminus of the protein.

The present invention provides novel fusion proteins useful for treatingcertain immune and inflammatory disorders. In the setting of autoimmune,alloimmune and inflammatory diseases, the fusion protein of thisinvention can reduce autoimmune, alloimmune and inflammatorymanifestations by one or more mechanisms. For example, the fusionprotein of this invention can bind to an activated immune cell, such asan activated T cell, that co-expresses on its surface a receptor for thefirst domain of the fusion protein, such as PD-1, as well as a receptorfor the second domain of the fusion protein, such as a TRAIL receptor ora Fas receptor. Through this binding event, the receptors for the firstdomain and for the second domain of the fusion protein may beco-triggered and thereby the death of the activated immune cell may beinduced.

Alternatively, the fusion protein of the present invention may mediateits activity by spanning two neighboring cells. For example, a PD-L1 orPD-L2 containing fusion protein can bind to the B7-1 costimulator on anantigen-presenting cell, thereby interfering with its costimulatory,immune-activating function. In addition, a PD-L1 or PD-L2-containingfusion protein can bind to PD-1 on an antigen-presenting cell andthereby elicit immunosuppressive cytokine production. Furthermore, onceanchored to a cell in one of these ways (that is, via binding to B7-1 orto PD-1), the apoptosis-inducing TRAIL ligand or Fas ligand, nowmembrane-anchored, can induce cell death in a neighboring activated Tcell, or, in the case of an antigen-presenting cell bearing receptorsfor TRAIL ligand or Fas ligand, autoinhibition (for example,auto-apoptosis) of the cell. Thus, the fusion proteins act to treatdisease by causing a reduction in certain immune cells.

Suitable first domains in the context of the PD-1 and TRAIL/Fassignaling axis include, for example, PD-L1 or PD-L2 protein itself,variants or derivatives of PD-L1 or PD-L2 protein, or other polypeptidesor proteins that are specifically designed to trigger the PD-1 receptor,such as agonistic anti-PD-1 Ab, and variants and/or derivatives ofthese. Preferably, the first domain of the fusion protein in thisembodiment comprises at least a portion of the extracellular domain ofPD-L1 or PD-L2 protein, specifically that portion which is necessary forbinding to a PD-1 receptor. Variants of the wild-type form of theextracellular domain of the PD-L 1 or PD-L2 protein, or the portion ofthe extracellular domain responsible for PD-1 receptor binding andtriggering, are also included in the present invention, so long as thevariant provides a similar level of biological activity as the wild-typeprotein.

Accordingly, the term “polypeptide that binds to and triggers PD-1receptor” as used herein includes, for example, PD-L1 or PD-L2 protein;the extracellular domain of PD-L1 or PD-L2 protein; a polypeptide whichcomprises at least a portion of the extracellular domain of PD-L1 orPD-L2 protein, which portion is responsible for binding to a PD-1receptor; antibodies to a PD-1 or B7-1 receptor; lipocalins engineeredto bind to a PD-1 receptor; and variants and/or derivatives of any ofthese. The term “PD-1” is understood to embrace polypeptidescorresponding to the complete amino acid sequence of the PD-1 protein,including the cytoplasmic, transmembrane and extracellular domains, aswell as polypeptides corresponding to smaller portions of the protein,such as the extracellular domain, or a portion of the extracellulardomain. In one embodiment the first domain of the PD-L1/TRAIL,PD-L2/TRAIL, PD-L1/FasL or PD-L2/FasL signaling pair comprises at leasta portion of the extracellular domain of the human PD-L1 or PD-L2protein.

PD-L1 and PD-L2 have two IgSF domains: one IgV and one IgC domain. Insome embodiments, the polypeptide that binds to and triggers PD-1receptor comprises at least 20 contiguous amino acids of the IgV domainof PD-L1 or PD-L2. In some embodiments, the polypeptide that binds toand triggers PD-1 receptor comprises the entire IgV domain of PD-L1 orPD-L2. In some embodiments, the polypeptide that binds to and triggersPD-1 receptor comprises at least 20 contiguous amino acids of the IgVand the IgC domain of PD-L1 or PD-L2. In some embodiments, thepolypeptide that binds to and triggers PD-1 receptor comprises theentire IgV and IgC domains of PD-L1 or PD-L2. The human PD-L1 IgV domaincovers amino acids 19-127 and the IgC domain amino acids covers aminoacids 153-210. The human PD-L2 IgV domain covers amino acids 21-118 andthe IgC domain covers amino acids 137-193.

Suitable second domains in the context of the PD-1/TRAIL signaling axisinclude, for example, the TRAIL protein itself, variants or derivativesof the TRAIL protein, or other polypeptides or proteins that arespecifically designed to inhibit activation of T cells or other cellsand/or induce apoptosis through binding to and triggering the TRAILreceptor, such as agonistic anti-TRAIL receptor Ab, and variants and/orderivatives of these. Preferably, the second domain of the fusionprotein in this embodiment comprises at least a portion of theextracellular domain of the TRAIL protein, specifically that portionwhich is necessary for binding to a TRAIL receptor. Variants of thewild-type form of the extracellular domain of the TRAIL protein, or theportion of the extracellular domain responsible for TRAIL receptorbinding and triggering, are also included in the present invention, solong as the variant provides a similar level of biological activity asthe wild-type protein.

Accordingly, the term “polypeptide that binds to a TRAIL receptor” asused herein includes, for example, the TRAIL protein; the extracellulardomain of the TRAIL protein; a polypeptide which comprises at least aportion of the extracellular domain of the TRAIL protein, which portionis responsible for binding to a TRAIL receptor; antibodies to a TRAILreceptor, or derivatives thereof; lipocalins engineered to bind to aTRAIL receptor; and variants and/or derivatives of any of these. Theterm “TRAIL” is understood to embrace polypeptides corresponding to thecomplete amino acid sequence of the TRAIL protein, including thecytoplasmic, transmembrane and extracellular domains, as well aspolypeptides corresponding to smaller portions of the protein, such asthe extracellular domain, or a portion of the extracellular domain. Inone embodiment the second domain of the PD-L1/TRAIL or PD-L2/TRAILsignaling pair comprises at least a portion of the extracellular domainof the human TRAIL protein.

In one embodiment, the fusion protein comprises a PD-L1/TRAIL fusionprotein. In an embodiment, the PD-L1/TRAIL fusion protein comprises thefusion protein having the amino acid sequence SEQ ID NO: 1. Theunderlined and bolded amino acids correspond to the second domain, whichdomain comprises a portion of TRAIL protein.

(SEQ ID NO: 1) MRIFAVFIFM TYWHLLNAFT VTVPKDLYVV EYGSNMTIECKFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSSYRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGGADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGYPKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIETIS   TVQEKQQNI SPLVRERGPQ   RVAAHITGTR   GRSNTLSSPN   SKNEKALGRKINSWESSRSG   HSFLSNLHLR   NGELVIHEKG   FYYIYSQTYF RFQEEIKENT  KNDKQMVQYI   YKYTSYPDPI   LLMKSARNSC WSKDAEYGLY   SIYQGGIFEL  KENDRIFVSV   TNEHLIDMDH EASFFGAFLV   G

In another embodiment, the PD-L1/TRAIL fusion protein comprises thefusion protein having the amino acid sequence SEQ ID NO: 2. Theunderlined and bolded amino acids correspond to the second domain, whichcomprises a portion of TRAIL protein.

(SEQ ID NO: 2) MRIFAVFIFM TYWHLLNAFT VTVPKDLYVV EYGSNMTIECKFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSSYRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGGADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGYPKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVIRGPQ   RVAAHITGTR GRSNTLSSPN   SKNEKALGRK   INSWESSRSG   HSFLSNLHLRNGELVIHEKG   FYYIYSQTYF   RFQEEIKENT   KNDKQMVQYI YKYTSYPDPI  LLMKSARNSC   WSKDAEYGLY   SIYQGGIFEL KENDRIFVSV   TNEHLIDMDH  EASFFGAFLV   G

In another embodiment, the PD-L1/TRAIL fusion protein comprises thefusion protein having the amino acid sequence SEQ ID NO: 3. Theunderlined and bolded amino acids correspond to the second domain, whichcomprises a portion of TRAIL protein. The bolded amino acids that arenot underlined correspond to a linker.

(SEQ ID NO: 3) MRIFAVFIFM TYWHLLNAFT VTVPKDLYVV EYGSNMTIECKFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSSYRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGGADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGYPKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRINTTTNEIFYCT FRRLDPEENH TAELVIGDPL VTAASVLEFG GSGGGSEGGG SEGGGSEGGG SDIETISTVQ   EKQQNISPLV RERGPQRVAA   HITGTRGRSN   TLSSPNSKNE   KALGRKINSWESSRSGHSFL   SNLHLRNGEL   VIHEKGFYYI   YSQTYFRFQE EIKENTKNDK  QMVQYIYKYT   SYPDPILLMK   SARNSCWSKD AEYGLYSIYQ   GGIFELKEND  RIFVSVTNEH   LIDMDHEASF FGAFLVG

In another embodiment, the PD-L1/TRAIL fusion protein comprises thefusion protein having the amino acid sequence SEQ ID NO: 4. Theunderlined and bolded amino acids correspond to the second domain, whichcomprises a portion of TRAIL protein. The bolded amino acids that arenot underlined correspond to a linker.

(SEQ ID NO: 4) MRIFAVFIFM TYWHLLNAFT VTVPKDLYVV EYGSNMTIECKFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSSYRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGGADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGYPKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRINTTTNEIFYCT FRRLDPEENH TAELVIGDPL VTAASVLEFG GSGGGSEGGG SEGGGSEGGG SDIRGPQRVA   AHITGTRGRS NTLSSPNSKN   EKALGRKINS   WESSRSGHSF   LSNLHLRNGELVIHEKGFYY   IYSQTYFRFQ   EEIKENTKND   KQMVQYIYKY TSYPDPILLM  KSARNSCWSK   DAEYGLYSIY   QGGIFELKEN DRIFVSVTNE   HLIDMDHEAS   FFGAFLVG

In one embodiment, the fusion protein comprises a PD-L2/TRAIL fusionprotein. In an embodiment, the PD-L2/TRAIL fusion protein comprises thefusion protein having the amino acid sequence SEQ ID NO: 5. Theunderlined and bolded amino acids correspond to the second domain, whichcomprises a portion of TRAIL protein.

(SEQ ID NO: 5) MIFLLLMLSL ELQLHQIAAL FTVTVPKELY IIEHGSNVTLECNFDTGSHV NLGAITASLQ KVENDTSPHR ERATLLEEQLPLGKASFHIP QVQVRDEGQY QCIIIYGVAW DYKYLTLKVKASYRKINTHI LKVPETDEVE LTCQATGYPL AEVSWPNVSVPANTSHSRTP EGLYQVTSVL RLKPPPGRNF SCVFWNTHVR ELTLASIDLQ SQMEPRTHPT ETISTVQEKQ   QNISPINRER GPQRVAAHIT   GTRGRSNTLS   SPNSKNEKAL  GRKINSWESS RSGHSFLSNL   HLRNGELVIH   EKGFYYIYSQ   TYFRFQEEIK ENTKNDKQMV  QYIYKYTSYP   DPILLMKSAR   NSCWSKDAEY GLYSIYQGGI   FELKENDRIF  VSVTNEHLID   MDHEASFFGA FLVG

In another embodiment, the PD-L2/TRAIL fusion protein comprises thefusion protein having the amino acid sequence SEQ ID NO: 6. Theunderlined and bolded amino acids correspond to the second domain, whichcomprises a portion of TRAIL protein.

(SEQ ID NO: 6) MIFLLLMLSL ELQLHQIAAL FTVTVPKELY IIEHGSNVTLECNFDTGSHV NLGAITASLQ KVENDTSPHR ERATLLEEQLPLGKASFHIP QVQVRDEGQY QCIIIYGVAW DYKYLTLKVKASYRKINTHI LKVPETDEVE LTCQATGYPL AEVSWPNVSVPANTSHSRTP EGLYQVTSVL RLKPPPGRNF SCVFWNTHVR ELTLASIDLQ SQMEPRTHPT RGPQRVAAHI   TGTRGRSNTL SSPNSKNEKA   LGRKINSWES   SRSGHSFLSN  LHLRNGELVI HEKGFYYIYS   QTYFRFQEEI   KENTKNDKQM   VQYIYKYTSY PDPILLMKSA  RNSCWSKDAE   YGLYSIYQGG   IFELKENDRI FVSVTNEHLI   DMDHEASFFG   AFLVG

In another embodiment, the PD-L2/TRAIL fusion protein comprises thefusion protein having the amino acid sequence SEQ ID NO: 7. Theunderlined and bolded amino acids correspond to the second domain, whichcomprises a portion of TRAIL protein. The bolded amino acids that arenot underlined correspond to a linker.

(SEQ ID NO: 7) MIFLLLMLSL ELQLHQIAAL FTVTVPKELY IIEHGSNVTLECNFDTGSHV NLGAITASLQ KVENDTSPHR ERATLLEEQLPLGKASFHIP QVQVRDEGQY QCIIIYGVAW DYKYLTLKVKASYRKINTHI LKVPETDEVE LTCQATGYPL AEVSWPNVSVPANTSHSRTP EGLYQVTSVL RLKPPPGRNF SCVFWNTHVRELTLASIDLQ SQMEPRTHPT GDPLVTAASV LEFGGSGGGS EGGGSEGGGS EGGGSDI ETI  STVQEKQQNI   SPLVRERGPQ RVAAHITGTR   GRSNTLSSPN   SKNEKALGRK  INSWESSRSG HSFLSNLHLR   NGELVIHEKG   FYYIYSQTYF   RFQEEIKENT KNDKQMVQYI  YKYTSYPDPI   LLMKSARNSC   WSKDAEYGLY SIYQGGIFEL   KENDRIFVSV  TNEHLIDMDH   EASFFGAFLV G

In another embodiment, the PD-L2/TRAIL fusion protein comprises thefusion protein having the amino acid sequence SEQ ID NO: 8. Theunderlined and bolded amino acids correspond to the second domain, whichcomprises a portion of TRAIL protein. The bolded amino acids that arenot underlined correspond to a linker.

(SEQ ID NO: 8) MIFLLLMLSL ELQLHQIAAL FTVTVPKELY IIEHGSNVTLECNFDTGSHV NLGAITASLQ KVENDTSPHR ERATLLEEQLPLGKASFHIP QVQVRDEGQY QCIIIYGVAW DYKYLTLKVKASYRKINTHI LKVPETDEVE LTCQATGYPL AEVSWPNVSVPANTSHSRTP EGLYQVTSVL RLKPPPGRNF SCVFWNTHVRELTLASIDLQ SQMEPRTHPT GDPLVTAASV LEFGGSGGGS EGGGSEGGGS EGGGSDI RGP  QRVAAHITGT   RGRSNTLSSP NSKNEKALGR   KINSWESSRS   GHSFLSNLHL  RNGELVIHEK GFYYIYSQTY   FRFQEEIKEN   TKNDKQMVQY   IYKYTSYPDP ILLMKSARNS  CWSKDAEYGL   YSIYQGGIFE   LKENDRIFVS VTNEHLIDMD   HEASFFGAFL   VG

The fusion proteins identified by the SEQ ID NOs listed above includeoriginal signal peptides; these signal peptides can be varied accordingto the needs of the user, the expression system, and other factors, aswould be understood by one skilled in the art. Signal peptides are wellknown in the art, and any desired signal peptide can be used, includingthose recognized/predicted by publicly available signal peptiderecognition software known to those skilled in the art.

In additional embodiments, the PD-L1/TRAIL fusion protein comprises avariant and/or derivative of the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. In further embodiments,the PD-L2/TRAIL fusion protein comprises a variant and/or derivative ofthe amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:7 or SEQ ID NO: 8.

Other suitable second domains in the context of the PD-1 and Fassignaling axis include, for example, the FasL protein itself, variantsor derivatives of the FasL protein, or other polypeptides or proteinsthat are specifically designed to inhibit activation of T cells or othercells and/or induce apoptosis through binding to and triggering the Fasreceptor, such as agonistic anti-Fas Ab, and variants and/or derivativesof these. Preferably, the second domain of the fusion protein in thisembodiment comprises at least a portion of the extracellular domain ofthe FasL protein, specifically that portion which is necessary forbinding to a Fas receptor. Variants of the wild-type form of theextracellular domain of the FasL protein, or the portion of theextracellular domain responsible for Fas receptor binding andtriggering, are also included in the present invention, so long as thevariant provides a similar level of biological activity as the wild-typeprotein.

Accordingly, the term “polypeptide that binds to a Fas receptor” as usedherein includes, for example, the FasL protein; the extracellular domainof the FasL protein; a polypeptide which comprises at least a portion ofthe extracellular domain of the FasL protein, which portion isresponsible for binding to a Fas receptor; antibodies to a Fas receptor;lipocalins engineered to bind to a Fas receptor; and variants and/orderivatives of any of these. The term “FasL” is understood to embracepolypeptides corresponding to the complete amino acid sequence of theFasL protein, including the cytoplasmic, transmembrane and extracellulardomains, as well as polypeptides corresponding to smaller portions ofthe protein, such as the extracellular domain, or a portion of theextracellular domain. In one embodiment the second domain of thePD-L1/FasL or PD-L2/FasL signaling pair comprises at least a portion ofthe extracellular domain of the human FasL protein.

In one embodiment, the fusion protein is a PD-L1/FasL fusion protein. Inan embodiment, the PD-L1/FasL fusion protein comprises the fusionprotein having the amino acid sequence SEQ ID NO:9. The underlined andbolded amino acids correspond to the second domain, which comprises aportion of FasL protein.

(SEQ ID NO: 9) MRIFAVFIFM TYWHLLNAFT VTVPKDLYVV EYGSNMTIECKFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSSYRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGGADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGYPKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH TAELVILEKQ   IGHPSPPPEK KELRKVAHLT   GKSNSRSMPL   EWEDTYGIVL   LSGVKYKKGGLVINETGLYF   VYSKVYFRGQ   SCNNLPLSHK   VYMRNSKYPQ DLVMMEGKMM  SYCTTGQMWA   RSSYLGAVFN   LTSADHLYVN VSELSLVNFE   ESQTFFGLYK   L

In an embodiment, the PD-L1/FasL fusion protein comprises the fusionprotein having the amino acid sequence SEQ ID NO: 10. The underlined andbolded amino acids correspond to the second domain which is a portion ofTRAIL protein. The bolded amino acids that are not underlined correspondto the linker.

(SEQ ID NO: 10) MRIFAVFIFM TYWHLLNAFT VTVPKDLYVV EYGSNMTIECKFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSSYRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGGADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGYPKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRINTTTNEIFYCT FRRLDPEENH TAELVIGDPL VTAASVLEFG GSGGGSEGGG SEGGGSEGGG SDILEKQIGH   PSPPPEKKEL RKVAHLTGKS   NSRSMPLEWE   DTYGIVLLSG   VKYKKGGLVINETGLYFVYS   KVYFRGQSCN   NLPLSHKVYM   RNSKYPQDLV MMEGKMMSYC  TTGQMWARSS   YLGAVFNLTS   ADHLYVNVSE LSLVNFEESQ   TFFGLYKL

In another embodiment, the fusion protein comprises a PD-L2/FasL fusionprotein. In an embodiment, the PD-L2/FasL fusion protein comprises afusion protein having the amino acid sequence SEQ ID NO:11. Theunderlined and bolded amino acids correspond to the second domain, whichcomprises a portion of FasL protein.

(SEQ ID NO: 11) MIFLLLMLSL ELQLHQIAAL FTVTVPKELY IIEHGSNVTLECNFDTGSHV NLGAITASLQ KVENDTSPHR ERATLLEEQLPLGKASFHIP QVQVRDEGQY QCIIIYGVAW DYKYLTLKVKASYRKINTHI LKVPETDEVE LTCQATGYPL AEVSWPNVSVPANTSHSRTP EGLYQVTSVL RLKPPPGRNF SCVFWNTHVR ELTLASIDLQ SQMEPRTHPT LEKQIGHPSP   PPEKKELRK V AHLTGKSNSR   SMPLEWEDTY   GIVLLSGVKY  KKGGLVINET GLYFVYSKVY   FRGQSCNNLP   LSHKVYMRNS   KYKIDLVMME GKMMSYCTTG  QMWARSSYLG   AVFNLTSADH   LYVNVSELSL VNFEESQTFF   GLYKL

In another embodiment, the PD-L2/FasL fusion protein comprises thefusion protein having the amino acid sequence SEQ ID NO: 12. Theunderlined and bolded amino acids correspond to the second domain, whichcomprises a portion of FasL protein. The bolded amino acids that are notunderlined correspond to a linker.

(SEQ ID NO: 12) MIFLLLMLSL ELQLHQIAAL FTVTVPKELY IIEHGSNVTLECNFDTGSHV NLGAITASLQ KVENDTSPHR ERATLLEEQLPLGKASFHIP QVQVRDEGQY QCIIIYGVAW DYKYLTLKVKASYRKINTHI LKVPETDEVE LTCQATGYPL AEVSWPNVSVPANTSHSRTP EGLYQVTSVL RLKPPPGRNF SCVFWNTHVRELTLASIDLQ SQMEPRTHPT GDPLVTAASV LEFGGSGGGS EGGGSEGGGS EGGGSDI LEK  QIGHPSPPPE   KKELRKVAHL TGKSNSRSMP   LEWEDTYGIV   LLSGVKYKKG  GLVINETGLY FVYSKVYFRG   QSCNNLPLSH   KVYMRNSKYP   QDLVMMEGKM MSYCTTGQMW  ARSSYLGAVF   NLTSADHLYV   NVSELSLVNF EESQTFFGLY   KL

SEQ ID NOs 9-12 include signal peptides; these signal peptides can bevaried according to the needs of the user, the expression system, andother factors, as would be understood by one skilled in the art. Signalpeptides are well known in the art, and any desired signal peptide canbe used, including those recognized/predicted by publicly availablesignal peptide recognition software known to those skilled in the art.

In additional embodiments, the PD-L1/FasL fusion protein comprises avariant and/or derivative of the amino acid sequence shown in SEQ IDNO:9 or SEQ ID NO:10. In further embodiments, the PD-L2/FasL fusionprotein is a variant and/or derivative of the amino acid sequence shownin SEQ ID NO:11 or SEQ ID NO:12.

In one embodiment, it is believed that the fusion proteins of thepresent invention inhibit activation of the immune system by preventingor reducing proliferation and differentiation of myelin-specific Tcells, or by inducing apoptosis those cells. In some embodiments thefusion proteins of the present invention may inhibit production ofpro-inflammatory cytokines and chemokines, such as IL-6, IL-8, RANTES,IP-10, and MCP-1, or inhibit potentiation of other cytokines/chemokines,such as TNF-α, and IL-1β; or inhibit induction of matrixmetalloproteinases such as MMP-1 and MMP-9; or inhibit prostaglandin E2secretion from fibroblasts and synoviocytes. The present inventionembraces inhibition/down-regulation of any and all cytokines that areeither promoted by PD-L1 or PD-L2 or down-modulated by the TRAIL ligand.

In other embodiments the fusion proteins of the present inventioninhibit autoreactive T cell proliferation, autoreactive antibodyproduction, and inflammatory reactions. In some embodiments, the fusionproteins of the present invention induce apoptosis in autoreactive Tcells.

In additional embodiments, the fusion proteins of the present inventionare believed to reduce inflammation as may be determined (i) in in vitroand in vivo assays that measure inhibition of pro-inflammatory cytokineand chemokine production and/or elevation of anti-inflammatory cytokineproduction; or (ii) in in vivo model systems of inflammation, such asautoimmune disease models, for example, EAE and collagen-inducedarthritis, and delayed-type hypersensitivity and other models in whichpro-inflammatory agents are introduced locally or systemically intoanimals. In these in vivo models, inflammation is assessed byhistological examination of inflamed tissues, isolation of inflammatorycells from diseased tissues, and measurement of disease manifestationsin affected animals. The fusion proteins of the present invention, inother embodiments, are believed to inhibit the proliferation,differentiation and/or effector function of pathogenic T cells such asautoreactive CD4+ T cells and CD8+ T cells and other pathogenic immunecells such as B cells, natural (NK) cells, NKT cells, lymphoidprogenitor cells, dendritic cells, monocytes/macrophages; induceapoptosis in pathogenic immune cells; promote generation of immune cellswith regulatory properties (such as CD4+ CD25+ regulatory T cells, Tr1cells, CD8+, NK NKT, and dendritic cells with immuno-inhibitoryactivities); decrease permeability of the blood-brain barrier, andthereby restrict access of inflammatory cells to the CNS; decreaseaccess of inflammatory cells to other disease sites, and decreaseangiogenesis associated with inflammation.

Most (although not all) of the TNF receptor (TNFR) superfamily membersare type II transmembrane proteins. These proteins contain anextracellular domain that is structurally characterized by the presenceof one to six cysteine-rich domains (CRDs). The typical CRD isapproximately 40 amino acids in length and contains six conservedcysteine residues that form three intrachain disulphide bridges. The CRDitself is typically composed of two distinct structural modules.

TRAIL

TRAIL is a Type II membrane protein having 281 amino acids and has beensequenced in a number of species, including, but not limited to, mouse:Swiss Prot. Accession No. P50592: human: Swiss Prot. Accession No.P50591; Rattus norvegicus: NCBI Accession NP—663714; Siniperca Chuatsi(Chinese Perch): NCBI Accession AAX77404; Gallus Gallus (Chicken): NCBIAccession BAC79267; Sus Scrofa (Pig): NCBI Accession NP—001019867;Ctenopharyngodon Idella (Grass Carp): NCBI Accession AAW22593; and BosTaurus (Cattle): NCBI Accession XP—001250249.

The extracellular domain of TRAIL comprises amino acids 39-281, and theTNF domain responsible for receptor binding comprises amino acids121-280, based on TNF homology models. The portion of the protein thatis particularly important for conferring activity has been identified.See, e.g., “Triggering cell death: The crystal structure of Apo2L/TRAILin a complex with death receptor”, Hymowitz S G, et al., Am. Mol. Cell.1999 October; 4(4):563-71), incorporated herein by reference, whichreports the most important amino acids for TRAIL binding to its receptorand activity are amino acids around the zinc area such as amino acids(191-201-205-207-236-237) and amino acids (150-216). See also, (1) KriegA et al 2003 Br. J of Cancer 88: 918-927, which describes two humanTRAIL variants without apoptotic activity, TRAIL-γ and TRAIL β; (2)“Enforced covalent trimerization increases the activity of the TNFligand family members TRAIL and CD95L”, D Berg et al., Cell death anddifferentiation (2007)14, 2021-2034; and (3) “Crystal Structure ofTRAIL-DR5 complex identifies a critical role of the unique frameinsertion in conferring recognition specificity”, S. Cha et al., J.Biol. Chem. 275: 31171-31177 (2000), all incorporated herein byreference.

TRAIL is known to ligate two types of receptors: death receptorstriggering TRAIL-induced apoptosis and decoy receptors that possiblyinhibit this pathway. Four human receptors for TRAIL have beenidentified: TRAILR1, TRAILR2, TRAILR3 and TRAILR4. TRAILR1 and TRAILR2when triggered induce apoptosis. However, TRAILR3 and TRAILR4 are decoyreceptors that do not induce apoptosis. TRAIL can also bind toosteoprotegrin (OPG). Binding to each of these receptors has beenwell-characterized, e.g., “The TRAIL apoptotic pathway in cancer onset,progression and therapy,” Nature Reviews Cancer Volume 8 (2008) 782-798.

Full-length human TRAIL (UniProtKG/Swiss-Prot accession number P50591.1)has the following amino acid sequence (SEQ ID NO:13). The extracellulardomain comprising amino acids 38-281 is underlined and in bold:

(SEQ ID NO: 13) MAMMEVQGGP SLGQTCVLIV IFTVLLQSLC VAVTYVYFT N ELKQMQDKYS  KSGIACFLKE   DDSYWDPNDE   ESMNSPCWQV KWQLRQLVRK   MILRTSEETI  STVQEKQQNI   SPLVRERGPQ RVAAHITGTR   GRSNTLSSPN   SKNEKALGRK  INSWESSRSG HSFLSNLHLR   NGELVIHEKG   FYYIYSQTYF   RFQEEIKENT KNDKQMVQYI  YKYTSYPDPI   LLMKSARNSC   WSKDAEYGLY SIYQGGIFEL   KENDRIFVSV  TNEHLIDMDH   EASFFGAFLV G

In some preferred embodiments, the second domain of the fusion proteincomprises the following amino acid sequence (SEQ ID NO:14) from humanTRAIL:

(SEQ ID NO: 14) ETI STVQEKQQNI SPLVRERGPQRVAAHITGTR GRSNTLSSPN SKNEKALGRK INSWESSRSGHSFLSNLHLR NGELVIHEKG FYYIYSQTYF RFQEEIKENTKNDKOMVQYI YKYTSYPDPI LLMKSARNSC WSKDAEYGLYSIYQGGIFEL KENDRIFVSV TNEHLIDMDH EASFFGAFLV G 

In some preferred embodiments, the second domain of the fusion proteincomprises the following amino acid sequence (SEQ ID NO:15) from humanTRAIL:

(SEQ ID NO: 15) RGPQ RVAAHITGTR GRSNTLSSPN SKNEKALGRK INSWESSRSGHSFLSNLHLR NGELVIHEKG FYYIYSQTYF RFQEEIKENTKNDKQMVQYI YKYTSYPDPI LLMKSARNSC WSKDAEYGLYSIYQGGIFEL KENDRIFVSV TNEHLIDMDH EASFFGAFLV G 

In some embodiments, the second domain of the fusion protein of theinvention comprises the extracellular domain of the human TRAIL protein.In other embodiments, the second domain of the fusion protein comprisesa fragment of TRAIL comprising at least 20, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 260, 270 or 280 contiguous amino acids of the full-lengthTRAIL protein, wherein the fragment binds and triggers TRAIL receptor.

Fas Ligand

Fas ligand (FasL) is a cytokine that binds to TNFRSF6/FAS, a receptorthat transduces the apoptotic signal into cells. It may be involved incytotoxic T-cell mediated apoptosis and in T-cell development.Fas-mediated apoptosis may have a role in the induction of peripheraltolerance, in the antigen-stimulated suicide of mature T cells, or both.Fas ligand has been sequenced in several species including human(UniProtKB/Swiss-Prot accession number P48023), mouse (GenBank accessionnumber AAA19778.1), horse (GenBank accession number ACV52391.1), cat(GenBank accession number BAC76426.1) and cattle (GenBank accessionnumber AEV59556.1).

The extracellular domain of Fas ligand comprises Fas ligand amino acids103-281. The cytoplasmic domain comprises amino acids 1-80, and thetransmembrane domain comprises amino acids 81-102.

The amino acid sequence of full-length human Fas ligand (FASLG; CD95L;FASL; TNFSF6) protein is shown below (SEQ ID NO:16)(UniProtKB/Swiss-Prot accession number P48023). The extracellulardomain, comprising amino acids 103-281, is underlined and in bold:

(SEQ ID NO: 16) MQQPFNYPYP QIYWVDSSAS SPWAPPGTVL PCPTSVPRRPGQRRPPPPPP PPPLPPPPPP PPLPPLPLPP LKKRGNHSTG LCLLVMFFMV LVALVGLGLG MFQLFHLQKE   LAELRESTSQ MHTASSLEKQ   IGHPSPPPEK   KELRKVAHLT   GKSNSRSMPLEWEDTYGIVL   LSGVKYKKGG   LVINETGLYF   VYSKVYFRGQ SCNNLPLSHK  VYMRNSKYPQ   DLVMMEGKMM   SYCTTGQMWA RSSYLGAVFN   LTSADHLYVN  VSELSLVNFE   ESQTFFGLYK L

In some preferred embodiments, the second domain of the fusion proteincomprises the following amino acid sequence (SEQ ID NO:17) from humanFasL:

(SEQ ID NO: 17) LEKQIGHPSPPPEK   KELRKVAHLT   GKSNSRSMPL EWEDTYGIVL  LSGVKYKKGG   LVINETGLYF   VYSKVYFRGQ SCNNLPLSHK   VYMRNSKYPQ  DLVMMEGKMM   SYCTTGQMWA RSSYLGAVFN   LTSADHLYVN   VSELSLVNFE  ESQTFFGLYK L

In some embodiments, the second domain of the fusion protein of theinvention comprises the extracellular domain of the human FasL protein.In other embodiments, the second domain of the fusion protein comprisesa fragment of FasL protein comprising at least 20, 30, 40, 50, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,230, 240, 250, 260, 270 or 280 contiguous amino acids of the full-lengthFasL protein, wherein fragment binds to and triggers Fas receptor.

PD-L1 and PD-L2

The signal peptide of PD-L1 comprises amino acids 1-17 or 1-18,depending on the source. The extracellular binding domain of PD-L1comprises amino acids 18-239 or 19-239, depending on the source.

The amino acid sequence of full-length human PD-L1 (B7-H1; PDCD1L1;PDL1; CD274) protein is shown below (SEQ ID NO:18) (UniProtKB/Swiss-Protaccession number Q9NZQ7.1). The extracellular domain, comprising aminoacids 18-239 or 19-239, depending on the source, is underlined and inbold:

(SEQ ID NO: 18) MRIFAVFIFM TYWHLLN AFT   VTVPKDLYVV   EYGSNMTIE CKFPVEKQLDL   AALIVYWEME   DKNIIQFVHG   EEDLKVQHSS YRQRARLLKD  QLSLGNAALQ   ITDVKLQDAG   VYRCMISYGG ADYKRITVKV   NAPYNKINQR  ILVVDPVTSE   HELTCOAEGY PKAEVIWTSS   DHQVLSGKTT   TTNSKREEKL  FNVTSTLRIN TTTNEIFYCT  FRRLDPEENH   TAELVI PELP LAHPPNERTHLVILGAILLC LGVALTFIFR LRKGRMMDVK KCGIQDTNSK KQSDTHLEET

In some embodiments, the first domain of the fusion protein of theinvention comprises the extracellular domain of the human PD-L1 protein.In other embodiments, the first domain of the fusion protein comprises afragment of PD-L1 comprising at least 20, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 260, 270 or 280 contiguous amino acids of the full-lengthPD-L1 protein, wherein the fragment binds to and triggers PD-1.

The signal peptide of PD-L2 comprises amino acids 1-18 or 1-19,depending on the source. The extracellular domain of PD-L2 comprisesamino acids 20-220.

The amino acid sequence of full-length human PD-L2 (B7-DC; PDCD1L2;PDL2) protein is shown below(UniProtKB/Swiss-Prot accession numberQ9BQ51) (SEQ ID NO:19). The extracellular domain, comprising amino acids20-220, is underlined and in bold:

(SEQ ID NO: 19 MIFLLLMLSL ELQLHQIAA L   FTVTVPKELY   IIEHGSNVTLECNFDTGSHV   NLGAITASLQ   KVENDTSPHR   ERATLLEEQL PLGKASFHIP  QVQVRDEGQY   QCIIIYGVAW   DYKYLTLKVK ASYRKINTHI   LKVPETDEVE  LTCQATGYPL   AEVSWPNVSV PANTSHSRTP   EGLYQVTSVL   RLKPPPGRNF  SCVFWNTHVR ELTLASIDLQ SQMEPRTHPT WLLHIFIPFCIIAFIFIATVIALRKQLCQK LYSSKDTTKR PVTITKREVN SAI

In the following PD-L1 amino acid sequence (SEQ ID NO:18), the IgV-likedomain is underlined and IgC-like domain is double underlined.

(SEQ ID NO: 18) MRIFAVFIFM TYWHLLNAFT VTVPKDLYVV EYGSNMTIECKFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSSYRQRARLLK DQLSLGNAAL QITDVKLQDA GVYRCMISYGGADYKRITVK VNAPYNKINQ RILVVDPVTS EHELTCQAEG YPKAEVIWTS SDHQVLSGKTTTTNSKREEK LFNVTSTLRI NTTTNEIFYC TFRRLDPEEN HTAELVIPEL PLAHPPNERTHLVILGAILL CLGVALTFIF RLRKGRMMD VKKCGIQDT NSKKQSDTH LEET

In the following PD-L2 amino acid sequence (SEQ ID NO:19), the IgV-likedomain is underlined and IgC-like domain is double underlined.

(SEQ ID NO: 19) MIFLLLMLSL ELQLHQIAAL FTVTVPKELY IIEHGSNVTLECNFDTGSHV NLGAITASLQ KVENDTSPHR ERATLLEEQLPLGKASFHIP QVQVRDEGQY QCIIIYGVAW DYKYLTLKVK ASYRKINTHI LKVPETDEVELTCQATGYPL AEVSWPNVSV PANTSHSRTP EGLYQVTSVL RLKPPPGRNF SCVFWNTHVRELTLASIDLQ SQMEPRTHPT WLLHIFIPFC IIAFIFIATVIALRKQLCQK LYSSKDTTKR PVTTTKREVN SAI

In some embodiments, the first domain of the fusion protein of theinvention comprises the extracellular domain of the PD-L2 protein. Inother embodiments, the first domain of the fusion protein comprises afragment of PD-L2 comprising at least 20, 30, 40, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230,240, 250, 260, 270 or 280 contiguous amino acids of the full-lengthPD-L2 protein, wherein the fragment binds to and triggers PD-1.

Linkers

The first and second domains of the fusion proteins of the invention maybe optionally connected via a linker. The residues for the linker may beselected from naturally occurring amino acids, non-naturally occurringamino acids, and modified amino acids. The linker will typically connectthe carboxy terminus of the first domain to the amino terminus of thesecond domain. The reverse is also possible, i.e., using the linker toconnect the carboxy terminus of the second domain to the amino terminusof the first domain. The linker may alter the distance between the twostructural components of the fusion protein, as well as alter theflexibility of this region. The linker may comprise any number of aminoacids. The linker may thus comprise, for example, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or moreamino acids. In some embodiments, the linker may be composed of from 3to 60 amino acid residues, from 3 to 40 amino acids, from 3 to 30 aminoacids, from 3 to 24 amino acids, from 3 to 18 amino acids, or from 3 to15 amino acids. The linker may comprise, for example, a repeatingsub-sequence of 2, 3, 4, 5 or more amino acid residues, comprising 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12 or more repeats of the sub-sequence.

Linkers may be naturally-occurring sequences or designed sequences.Peptide linkers useful in the molecule of the invention include, but arenot limited to, glycine linkers, glycine-rich linkers, serine-glycinelinkers, and the like. A glycine-rich linker comprises at least about50% glycine and preferably at least about 60% glycine. In oneembodiment, the linker comprises the amino acid sequence Gly-Ser, orrepeats thereof. See, e.g., Huston, et al., Methods in Enzymology,203:46-88 (1991). In another embodiment, the linker comprises the aminoacid sequence Glu-Lys, or repeats thereof. See, e.g., Whitlow et al.,Protein Eng., 6:989 (1993)). In another embodiment, the linker comprisesthe amino acid sequence Gly-Gly-Ser, or repeats thereof. In anotherembodiment, the linker comprises the amino acid sequenceGly-Gly-Gly-Gly-Ser (SEQ ID NO:20), or repeats thereof. In certainspecific embodiments, the linker contains from 2 to 12 repeats ofGly-Gly-Ser or Gly-Gly-Gly-Gly-Ser (SEQ ID NO:21). See U.S. Pat. No.6,541,219 for examples of peptide linkers. In another embodiment, thelinker comprises the amino acid sequence of SEQ ID NO: 22:GDPLVTAASVLEFGGSGGGSEGGGSEGGGSEGGGSDI.

Linkers comprising human immunoglobulin Fe region sequences are alsouseful. An exemplary Fc region linker includes but is not limited to:the hinge region of human IgG1 (EPKSCDKTHTCPPCP; SEQ ID NO:23); theC_(H2) and C_(H3) domains of a human IgG1; and a second IgG1 hingeregion. An exemplary sequence for this linker comprises (SEQ ID NO: 24).The hinge region sequences are underlined.

(SEQ ID NO: 24) EPKSCDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQYNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKTISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPSDIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKSRWQQGNVFSC SVMHEALHNH YTQKSLSLSP GKEPKSCDKT HTCPPCP

In another embodiment, the hinge region and the C_(H2) and C_(H3)domains of human IgG1 are mutated to prevent inter-chain disulfidebonds, to reduce antibody dependent cellular cytotoxicity (ADCC), or toeliminate N-linked glycosylation (aglycosyl human IgG1). An exemplarysequence for this linker comprises the sequence below, wherein mutatedsequences are in bold and underlined.

(SEQ ID NO: 25) EPKS S DKTHT  S PP S PAP PVA  G A PSVFLFPP KPKDTLMISRTPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQYASTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKTISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPSDIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKSRWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK

Linkers are useful for separating the two components of the fusionprotein to enable proper folding of the components, to reduce potentialsteric problems, and/or to contribute optimal receptor binding. Theskilled artisan is familiar with the design and selection of peptidelinkers. See, for instance, Robinson et al., 1998, Proc. Natl. Acad.Sci. USA 95:5929-5934. Automated programs are also available for peptidelinker design (e.g., Crasto et al., 2000, Protein Engineering13:309-312).

Optional Other Elements

The fusion protein optionally may also include further elements apartfrom the first domain, the second domain and the optional linker. Suchfurther elements may include: an initiator methionine, a signal peptide,a trimerization domain, an antigen polypeptide, and a purification tag,such as His-6. Fusion proteins essentially consisting of the firstdomain and the second domain and an optional linker are preferredembodiments in the context of the present invention.

Fusion proteins of the invention optionally comprise a signal peptide.Signal peptides can be varied according to the needs of the user, theexpression system, and other factors, as would be understood by oneskilled in the art. Signal peptides are well known in the art, and anydesired signal peptide can be used, including those recognized/predictedby publicly available signal peptide recognition software known to thoseskilled in the art.

TRAIL and FasL both require trimerization for optimal receptor binding.Naturally-occurring TRAIL and FasL each can form trimers, however, thetrimers can be unstable. Thus, addition of a heterologous trimerizationdomain to TRAIL and FasL may further increase receptor binding affinityby increasing the likelihood of formation and stabilization of theresulting protein. Thus, in an embodiment, the fusion protein of theinvention optionally further comprises a heterologous trimerizationdomain. Within the fusion protein, the heterologous trimerization domainmay be positioned anywhere within the fusion protein, provided it doesnot disrupt the functional activity of the fusion protein, e.g., bindingto and triggering PD-1 and TRAIL receptor or Fas receptor. For instance,the trimerization domain should not disrupt the IgSF domain of PD-L1 orPD-L2 that underlies binding to PD-1. Similarly, the heterologoustrimerization domain should not disrupt the binding and triggeringfunction of the TRAIL or FasL domain. The heterologous trimerizationdomain may be positioned within the first domain outside of its IgSFdomains, or between the first domain and the optional linker, or withinor in place of the optional linker, or between the optional linker andthe second domain, or at the C-terminal terminus of the fusion protein.It is preferable that the heterologous trimerization domain ispositioned substantially adjacent to the second domain, to optimize theformation of the second domain trimers. In a preferred embodiment,however, the trimerization domain is not positioned at the C-terminalterminus of the fusion protein.

Trimerization domains are well known in the art. Non-limiting examplesof trimerization domains suitable as a heterologous trimerization domainin the fusion protein of the invention include: the GCN4 leucine zipper(Harbury et al., 1993, “A switch between two-, three-, and four-strandedcoiled coils in GCN4 leucine zipper mutants,” Science 262(5138):1401-7);a 35 amino-acid sequence from lung surfactant protein (Hoppe et al.,1994, “A parallel three stranded alpha-helical bundle at the nucleationsite of collagen triple-helix formation,” FEBS Lett. 344(2-3):191-5);short, repeating heptad sequences from collagen (McAlinden et al., 2003,“Alpha-helical coiled-coil oligomerization domains are almost ubiquitousin the collagen superfamily,” J Biol Chem. 278(43):42200-7. Epub 2003Aug. 14.); and the bacteriophage T4 fibritin “foldon” (see, e.g.,Miroshnikov et al., 1998, “Engineering trimeric fibrous proteins basedon bacteriophage T4 adhesins,” Protein Eng. 11(4):329-32). Exemplarytrimerization domains are also disclosed in U.S. Pat. Nos. 6,911,205 and8,147,843, and U.S. Pat. Appln. Pub. 2010/0136032. An exemplarytrimerization sequence is the T4 “foldon” having the sequence:GYIPEAPRDGQAYVRKRGEWVLLSTFL (SEQ ID NO: 26). Another exemplarytrimerization domain is from thrombospondin-1 and has the sequence:VTTLQDSIRKVTEENKELANELRR (SEQ ID NO: 27).

Modifications

Additional modifications can be introduced such as those that furtherstabilize the TRAIL trimer and/or increase affinity of binding to theTRAIL receptor, or stabilize the Fas L trimer and/or increase affinityof binding to Fas receptor; and spacers/linkers can be added to alterthe distance between the two structural components of the fusionprotein, as well as alter the flexibility of this region.

In additional embodiments, the fusion proteins of the present inventionmay further comprise one or more additional polypeptide domains added tofacilitate protein purification, to increase expression of therecombinant protein, or to increase the solubility of the recombinantprotein. Such purification/expression/solubility facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilisedmetals (Porath J (1992) Protein Expr Purif 3-.26328 1), protein Adomains that allow purification on immobilised immunoglobulin, and thedomain utilised in the FLAGS extension/affinity purification system(Immunex Corp, Seattle, Wash.). The inclusion of a cleavable linkersequence such as Factor Xa or enterokinase (Invitrogen, San Diego,Calif.) between the purification domain and the fusion proteins of thepresent invention is useful to facilitate purification.

This invention relates to PD-L1/TRAIL, PD-L2/TRAIL, PD-L1/FasL,PD-L2/FasL and related fusion proteins. The invention also encompassesvariants of the fusion proteins. While in general it is desirable forvariants to show enhanced ability for binding to a given molecule, insome embodiments variants may be designed with slightly reduced activityas compared to other fusion proteins of the invention, for example, ininstances in which one would purposefully want to attenuate activity.Moreover, variants or derivatives can be generated that would bind moreselectively to one of the TRAIL receptor variants (there are three TRAILreceptors in humans). Furthermore, variants or derivatives can begenerated that would have altered multimerization properties. Whenengineering variants, this could be done for either the entire TRAILextracellular domain, or for that component of the extracellular domainthat is incorporated within the fusion protein itself.

Preferably, variants or derivatives of the fusion proteins of thepresent invention maintain the hydrophobicity/hydrophilicity of theamino acid sequence.

The invention also provides chemical modification of a fusion protein ofthe invention. Non-limiting examples of such modifications may includebut are not limited to aliphatic esters or amides of the carboxylterminus or of residues containing carboxyl side chains, O-acylderivatives of hydroxyl group-containing residues, and N-acylderivatives of the amino-terminal amino acid or amino-group containingresidues, e.g., lysine or arginine.

Additional modifications can include, for example, production of afusion protein conjugated with polyethylene glycol (PEG), or addition ofPEG during chemical synthesis of a polypeptide of the invention.Modifications of polypeptides or portions thereof can also includereduction/alkylation; chemical coupling to an appropriate carrier ormild formalin treatment.

Other derivatives of the fusion proteins of the present inventioninclude incorporation of unnatural amino acid residues, orphosphorylated amino acid residues such as phosphotyrosine,phosphoserine or phosphothreonine residues. Other potentialmodifications include sulfonation, biotinylation, or the addition ofother moieties, particularly those which have molecular shapes similarto phosphate groups.

Derivatives also include polypeptides modified by glycosylation. Thesecan be made by modifying glycosylation patterns during synthesis andprocessing in various alternative eukaryotic host expression systems, orduring further processing steps. Methods for producing glycosylationmodifications include exposing the fusion proteins to glycosylatingenzymes derived from cells that normally carry out such processing, suchas mammalian glycosylation enzymes. Alternatively, deglycosylationenzymes can be used to remove carbohydrates attached during productionin eukaryotic expression systems. Additionally, one can also modify thecoding sequence so that glycosylation site(s) are added or glycosylationsites are deleted or disabled. Furthermore, if no glycosylation isdesired, the proteins can be produced in a prokaryotic host expressionsystem.

Variants and/or derivatives of the fusion proteins of the invention canbe prepared by chemical synthesis or by using site-directed mutagenesis(Gillman et al., Gene 8:81 (1979); Roberts et al., Nature 328:731 (1987)or Innis (Ed.), 1990, PCR Protocols: A Guide to Methods andApplications, Academic Press, New York, N.Y.) or the polymerase chainreaction method (PCR; Saiki et al., Science 239:487 (1988)), asexemplified by Daugherty et al. (Nucleic Acids Res. 19:2471 (1991)) tomodify nucleic acids encoding the complete receptors.

Additional modifications can be introduced such as those that furtherstabilize the TRAIL trimer and/or increase affinity of binding to theTRAIL receptor. In additional embodiments, the fusion proteins of thepresent invention may further comprise one or more additionalpolypeptide domains added to facilitate protein purification, toincrease expression of the recombinant protein, or to increase thesolubility of the recombinant protein. Suchpurification/expression/solubility facilitating domains include, but arenot limited to, metal chelating peptides such as histidine-tryptophanmodules that allow purification on immobilized metals (Porath J (1992)Protein Expr Purif 3-0.26328 1), protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAGS extension/affinity purification system (Immunex Corp, Seattle,Wash.). The inclusion of a cleavable linker sequence such as Factor Xaor enterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and PD-L1/TRAIL, PD-L2/TRAIL, PD-L1/FasL or PD-L2/FasL is usefulto facilitate purification.

PD-L1 and PD-L2 are monomeric. However, once linked to second domaincomponents TRAIL or FasL which are naturally trimeric, a de facto PD-L1or PD-L2 trimer is formed. This serves to bring PD-1 receptors intoproximity, likely enhancing their triggering. Additionally, higher-ordercomplexes may result, such as hexamers, which are formed for example inthe case of FasL when engaged with its receptor Fas.

In another embodiment a fusion protein of the present invention maycontain a heterologous signal sequence at its N-terminus. In certainhost cells (e.g., mammalian host cells), expression and/or secretion ofthe fusion protein can be increased through use of a heterologous signalsequence. Signal sequences are typically characterized by a core ofhydrophobic amino acids, which are generally cleaved from the matureprotein during secretion in one or more cleavage events. Such signalpeptides contain processing sites that allow cleavage of the signalsequence from the mature proteins as they pass through the secretorypathway. Thus, the invention pertains to the described polypeptideshaving a signal sequence, as well as to polypeptides from which thesignal sequence has been proteolytically cleaved (i.e., the cleavageproducts).

In order to enhance stability and/or reactivity, the fusion proteins ofthe present invention can also be modified to incorporate one or morepolymorphisms in the amino acid sequence resulting from natural allelicvariation. Additionally, D-amino acids, non-natural amino acids ornon-amino acid analogues can be substituted or added to produce amodified fusion protein within the scope of this invention.

Expression of Fusion Proteins

The amino acid sequences of the present invention may be produced byexpression of a nucleotide sequence coding for same in a suitableexpression system.

Fusion protein expression vectors include pGEX (Pharmaci, a Piscataway,N.J.), pMAL (New England Biolabs, Beverly, Mass.) and pRITS (Pharmacia,Piscataway, N.J.) which fuse glutathione S transferase (GST), maltose Bbinding protein, or protein A, respectively, to the target recombinantprotein. EBV, BKV, and other episomal expression vectors (Invitrogen)can also be used. In addition, retroviral and lentiviral expressionvectors can also be used. Furthermore, any one of a number of in vivoexpression systems designed for high level expression of recombinantproteins within organisms can be invoked for producing the fusionproteins specified herein.

In addition, or in the alternative, the fusion protein itself can beproduced using chemical methods to synthesize the desired amino acidsequence, in whole or in part. For example, polypeptides can besynthesized by solid phase techniques, cleaved from the resin, andpurified by preparative high performance liquid chromatography (e.g.,Creighton (1983) Proteins Structures And Molecular Principles, WHFreeman and Co, New York N.Y.). The composition of the syntheticpolypeptides may be confirmed by amino acid analysis or sequencing(e.g., the Edman degradation procedure). Additionally, the amino acidsequence of a fusion protein of the invention, or any part thereof, maybe altered during direct synthesis and/or combined using chemicalmethods with a sequence from other subunits, or any part thereof, toproduce a variant polypeptide.

Assays For Fusion Protein Activity

Any of the various immunologic assays known in the art may be used tomeasure the immunologic activity of any fusion protein.

For example, any one of several conventional assays for monitoringcytokine production, as a measure of immune cells activation anddifferentiation, can be invoked. For example, for tracking T cellactivation, interleukin-2 can be employed as a marker, which can beassayed as described in Thompson C B, et al., Proc. Natl. Acad. Sci.USA. 86:1333 (1989) the entire disclosure of which is incorporatedherein by reference. A kit for an assay for the production of interferonis also available from Genzyme Corporation (Cambridge, Mass.). One canalso employ immunofluorescence and flow cytometry to monitor cytokineproduction on a cellular basis, and to monitor cell surface markers thatreflect cellular activation and/or differentiation states. A host ofsuch markers are known, detecting antibodies are broadly commerciallyavailable, and the markers are well known in the art.

A common assay for T cell proliferation entails measuring tritiatedthymidine incorporation. The proliferation of T cells can be measured invitro by determining the amount of 3H-labeled thymidine incorporatedinto the replicating DNA of cultured cells. Therefore, the rate of DNAsynthesis and, in turn, the rate of cell division can be quantified.

Another assay for monitoring T cell proliferation is based on loading Tcells with the CFSE dye, and subsequently monitoring by flow cytometrythe dilution of this dye that accompanies successive cell divisions. Inaddition to monitoring the inhibition of T cell proliferation, thebioactivity of the fusion protein can also be monitored by evaluatingits capacity to induce apoptosis in TRAIL receptor-positive tumor celllines in which TRAIL receptor triggering leads to apoptosis. Bycombining these cells with other cells that have PD-1 on their surfaces,one can assess whether new fusion protein derivatives both anchor toPD-1 and thereby have their pro-apoptotic TRAIL-driven activity enhancedin this way.

Pharmaceutical Compositions, Dosing Regimens and Treatment Methods

Administration of the compositions of this invention is typicallyparenteral, such as by subcutaneous, intravenous, intramuscular, orintraperitoneal injection, or by infusion or by any other acceptablesystemic method. Administration by subcutaneous injection is a preferredembodiment. An alternative route of administration is by intravenousinfusion, which may typically take place over a time course of about 1to 5 hours. In addition, there are a variety of oral delivery methodsfor administration of therapeutic proteins, and these can be applied tothe therapeutic fusion proteins of this invention.

Often, treatment dosages are titrated upward from a low level tooptimize safety and efficacy. Generally, daily dosages will fall withina range of about 0.01 to 20 mg protein per kilogram of body weight.Typically, the dosage range will be from about 0.1 to 5 mg protein perkilogram of body weight. Various modifications or derivatives of thefusion proteins, such as addition of polyethylene glycol chains(PEGylation), may be made to influence their pharmacokinetic and/orpharmacodynamic properties.

To administer the fusion protein by other than parenteraladministration, it may be necessary to coat the protein with, orco-administer the protein with, a material to prevent its inactivation.For example, protein may be administered in an incomplete adjuvant,co-administered with enzyme inhibitors or in liposomes. Enzymeinhibitors include pancreatic trypsin inhibitor,diisopropylfluorophosphate (DEP) and trasylol. Liposomes includewater-in-oil-in-water CGF emulsions as well as conventional liposomes(Strejan et al., (1984) J. Neuroimmunol. 7:27).

The amount of the fusion protein of the invention administered will bean amount that will ameliorate one or more of the well-known parametersthat characterize medical conditions caused by autoimmune disease,alloimmune disease, inflammatory disease or cancer. One such autoimmunedisease is multiple sclerosis, for example. Many such parameters andconditions have been described. An effective amount, in the context ofmultiple sclerosis, will be the amount of fusion protein that issufficient to accomplish one or more of the following: decrease theseverity of symptoms; decrease the duration of disease exacerbations;increase the frequency and duration of disease remission/symptom-freeperiods; prevent fixed impairment and disability; and/orprevent/attenuate chronic progression of the disease. Clinically, thiswould result in improvement in visual symptoms (visual loss, diplopia),gait disorders (weakness, axial instability, sensory loss, spasticity,hyperreflexia, loss of dexterity), upper extremity dysfunction(weakness, spasticity, sensory loss), bladder dysfunction (urgency,incontinence, hesitancy, incomplete emptying), depression, emotionallability, and cognitive impairment. Pathologically the treatment withfusion proteins of the present invention is believed to be capable ofone or more of the following, such as myelin loss, breakdown of theblood-brain barrier, perivascular infiltration of mononuclear cells,immunologic abnormalities, gliotic scar formation and astrocyteproliferation, metalloproteinase production, and impaired conductionvelocity.

Although the compositions of this invention can be administered insimple solution, they are more typically used in combination with othermaterials such as carriers, preferably pharmaceutical carriers. Usefulpharmaceutical carriers can be any compatible, non-toxic substancesuitable for delivering the compositions of the invention to a patient.Sterile water, alcohol, fats, waxes, and inert solids may be included ina carrier. Pharmaceutically acceptable adjuvants (buffering agents,dispersing agents) may also be incorporated into the pharmaceuticalcomposition. Generally, compositions useful for parenteraladministration of such drugs are well known; e.g. Remington'sPharmaceutical Science, 17th Ed. (Mack Publishing Company, Easton, Pa.,1990). Alternatively, compositions of the invention may be introducedinto a patient's body by implantable drug delivery systems (Urquhart etal., Ann. Rev. Pharmacol. Toxicol. 24:199 (1984)).

Therapeutic formulations may be administered in many conventional dosageformulations. Formulations typically comprise at least one activeingredient, together with one or more pharmaceutically acceptablecarriers. Formulations may include those suitable for oral, rectal,nasal, or parenteral (including subcutaneous, intramuscular, intravenousand intradermal) administration.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. See,e.g., Gilman et al. (eds.) (1990), The Pharmacological Bases ofTherapeutics, 8th Ed., Pergamon Press; and Remington's PharmaceuticalSciences, supra, Easton, Pa.; Avis et al. (eds.) (1993) PharmaceuticalDosage Forms: Parenteral Medications Dekker, N.Y.; Lieberman et al.(eds.) (1990) Pharmaceutical Dosage Forms: Tablets Dekker, N.Y.; andLieberman et al. (eds.) (1990), Pharmaceutical Dosage Forms: DisperseSystems Dekker, N.Y.

In additional embodiments, the present invention contemplatesadministration of the fusion proteins by gene therapy methods, e.g.,administration of an isolated nucleic acid encoding a fusion protein ofinterest. The protein building blocks (e.g., first and second domains)of the fusion proteins of the present invention have beenwell-characterized, both as to the nucleic acid sequences encoding theproteins and the resultant amino acid sequences of the proteins.Engineering of such isolated nucleic acids by recombinant DNA methods iswell within the ability of one skilled in the art. Codon optimization,for purposes of maximizing recombinant protein yields in particular cellbackgrounds, is also well within the ability of one skilled in the art.Administration of an isolated nucleic acid encoding the fusion proteinis encompassed by the expression “administering a therapeuticallyeffective amount of a fusion protein”. Gene therapy methods are wellknown in the art. See, e.g., WO96/07321 which discloses the use of genetherapy methods to generate intracellular antibodies. Gene therapymethods have also been successfully demonstrated in human patients. See,e.g., Baumgartner et al., Circulation 97: 12, 1114-1123 (1998), and morerecently, Fatham, C. G. ‘A gene therapy approach to treatment ofautoimmune diseases’, Immun. Res. 18:15-26 (2007); and U.S. Pat. No.7,378,089, both incorporated herein by reference. See also Bainbridge JW B et al. “Effect of gene therapy on visual function in Leber'scongenital Amaurosis”. N Engl Med 358:2231-2239, 2008; and Maguire A Met al. “Safety and efficacy of gene transfer for Leber's. CongenitalAmaurosis”. N Engl J Med 358:2240-8, 2008.

There are two major approaches for introducing a nucleic acid encodingthe fusion protein (optionally contained in a vector) into a patientscells; in vivo and ex vivo. For in vivo delivery the nucleic acid isinjected directly into the patient, usually at the site where the fusionprotein is required. For ex vivo treatment, the patient's cells areremoved, the nucleic acid is introduced into these isolated cells andthe modified cells are administered to the patient either directly or,for example, encapsulated within porous membranes which are implantedinto the patient (see, e.g., U.S. Pat. Nos. 4,892,538 and 5,283,187).There are a variety of techniques available for introducing nucleicacids into viable cells. The techniques vary depending upon whether thenucleic acid is transferred into cultured cells in vitro, or in vivo inthe cells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, the calciumphosphate precipitation method, etc. Commonly used vectors for ex vivodelivery of the gene are retroviral and lentiviral vectors.

Preferred in vivo nucleic acid transfer techniques include transfectionwith viral vectors such as adenovirus, Herpes simplex I virus,adeno-associated virus; lipid-based systems (useful lipids forlipid-mediated transfer of the gene are DOTMA, DOPE and DC-Chol, forexample); naked DNA; and transposon-based expression systems. For areview of gene marking and gene therapy protocols, see Anderson et al.,Science 256:808-813 (1992). See also WO 93/25673 and the referencescited therein.

“Gene therapy” includes both conventional gene therapy where a lastingeffect is achieved by a single treatment, and the administration of genetherapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA.Oligonucleotides can be modified to enhance their uptake, e.g. bysubstituting their negatively charged phosphodiester groups by unchargedgroups. The fusion proteins of the present invention can be deliveredusing gene therapy methods, for example locally in tumor beds,intrathecally, or systemically (e.g., via vectors that selectivelytarget specific tissue types, for example, tissue-specificadeno-associated viral vectors). In some embodiments, primary cells(such as lymphocytes or stem cells) from the individual can betransfected ex vivo with a gene encoding any of the fusion proteins ofthe present invention, and then returning the transfected cells to theindividual's body.

In some embodiments, the fusion proteins of the present invention aresuitable for treatment of immune system diseases or disorders,including, but not limited to, idiopathic thrombocytopenia purpura,autoimmune neutropenia, autoimmunocytopenia, antiphospholipid syndrome,gluten-sensitive enteropathy, allergic encephalomyelitis, myocarditis,relapsing polychondritis, rheumatic heart disease, neuritis, uveitisophthalmia, polyendo-crinopathies, purpura (e.g., Henloch-Scoenleinpurpura), Reiter's Disease, Stiff-Man Syndrome, autoimmune pulmonaryinflammation, myocarditis, IgA glomerulonephritis, dense depositdisease, rheumatic heart disease, Guillain-Barre Syndrome, insulindependent diabetes mellitus, autoimmune inflammatory eye, autoimmunethyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis), discoidlupus, Goodpasture's syndrome, pemphigus, receptor autoimmunities (suchas, for example, Graves' Disease and insulin resistance), autoimmunethrombocytopenic purpura, rheumatoid arthritis, schleroderma withanti-collagen antibodies, mixed connective tissue disease,polymyositis/dermatomyositis, pernicious anemia, idiopathic Addison'sdisease, infertility, glomerulonephritis such as primaryglomerulonephritis and IgA nephropathy, bullous pemphigoid, Sjogren'ssyndrome, diabetes mellitus, and adrenergic drug resistance (includingadrenergic drug resistance with asthma or cystic fibrosis), chronicactive hepatitis, primary biliary cirrhosis, other endocrine glandfailure, vitiligo, vasculitis, post-MI cardiotomy syndrome, urticaria,atopic dermatitis, asthma, inflammatory myopathies, and otherinflammatory, granulomatous, degenerative, and atrophic disorders),atherosclerosis and epilepsy.

Specific autoimmune disorders that may be ameliorated using thecompounds and methods of this invention include without limitation,autoimmune disorders of the nervous system (e.g., multiple sclerosis,myasthenia gravis, autoimmune neuropathies such as Guillain-Barre, andautoimmune uveitis), autoimmune disorders of the blood (e.g., autoimmunehemolytic anemia, pernicious anemia, and autoimmune thrombocytopenia),autoimmune disorders of the blood vessels (e.g., temporal arteritis,anti-phospholipid syndrome, vasculitides such as Wegener'sgranulomatosis, and Behcet's disease), autoimmune disorders of the skin(e.g., psoriasis, dermatitis herpetiformis, pemphigus vulgaris, andvitiligo), autoimmune disorders of the gastrointestinal system (e.g.,Crohn's disease, ulcerative colitis, primary biliary cirrhosis, andautoimmune hepatitis), autoimmune disorders of the endocrine glands(e.g., Type 1 or immune-mediated diabetes mellitus, Grave's disease,Hashimoto's thyroiditis, autoimmune oophoritis and orchitis, andautoimmune disorder of the adrenal gland); and autoimmune disorders ofmultiple organs (including connective tissue and musculoskeletal systemdiseases) (e.g., rheumatoid arthritis, systemic lupus erythematosus,scleroderma, polymyositis, dermatomyositis, spondyloarthropathies suchas ankylosing spondylitis, and Sjogren's syndrome).

Examples of inflammatory disorders include chronic inflammatorydisorders of the joints, including arthritis, osteoarthritis and bonediseases associated with increased bone resorption; inflammatory boweldiseases such as ileitis, Barrett's syndrome; inflammatory lungdisorders such as asthma, adult respiratory distress syndrome, andchronic obstructive airway disease; inflammatory disorders of the eyeincluding corneal dystrophy, trachoma, onchocerciasis, uveitis,sympathetic ophthalmitis and endophthalmitis; chronic inflammatorydisorders of the gums, including gingivitis and periodontitis;tuberculosis; leprosy; inflammatory diseases of the kidney includinguremic complications, glomerulonephritis and nephrosis; inflammatorydisorders of the skin including sclerodermatitis, psoriasis and eczema;inflammatory diseases of the central nervous system, including chronicdemyelinating diseases of the nervous system, multiple sclerosis,AIDS-related neurodegeneration and Alzheimer's disease, infectiousmeningitis, encephalomyelitis, Parkinson's disease, Huntington'sdisease, amyotrophic lateral sclerosis and viral or autoimmuneencephalitis; autoimmune disorders, immune-complex vasculitis, systemiclupus and erythematodes; systemic lupus erythematosus (SLE); andinflammatory diseases of the heart such as cardiomyopathy, ischemicheart disease hypercholesterolemia, atherosclerosis; as well as variousother diseases with significant inflammatory components, includingpreeclampsia; chronic liver failure, brain and spinal cord trauma. Theremay also be a systemic inflammation of the body, exemplified bygram-positive or gram negative shock, hemorrhagic or anaphylactic shock,or shock induced by cancer chemotherapy in response to pro-inflammatorycytokines, e.g., shock associated with pro-inflammatory cytokines. Suchshock can be induced, e.g., by a chemotherapeutic agent used in cancerchemotherapy.

Because a number of immune disorders are caused by inflammation, thereis some overlap between disorders that are considered immune diseasesand inflammatory diseases. For the purpose of this disclosure, in thecase of such an overlapping disorder, the disorder may be consideredeither an immune disease or an inflammatory disease.

In one embodiment, the fusion proteins of the present invention are usedto treat multiple sclerosis.

Mast cells are known to act as antigen-presenting cells and also expressPD-L2. In certain pathologic circumstances, it is contemplated that mastcells may also neo-express PD-1. An allergic disorder may include mastcells the express PD-1. Administration of a fusion protein of theinvention is contemplated as a therapeutic for such a disorder. Thus, inanother embodiment, the fusion proteins of the present invention areused to treat a disorder, the pathology of which pathology includes mastcells that express PD-1. It is also known that T cells play a role inmolding mast cell response. Accordingly, it is further contemplated thatthe fusion protein of the invention may achieve efficacy in diseasesincluding mast cell pathology by an indirect effect, via the effect ofthe fusion protein on T cell subsets that interface with the pathogenicmast cells in allergic disorders.

In additional embodiments, the fusion proteins of the present inventioncan be used to treat various types of cancer. Soluble TRAIL has beenassociated with the induction of apoptosis in certain kinds of tumorcells. Moreover, for certain tumor types, inflammation may actually bepro-tumorigenic. Hence, a TRAIL fusion protein can be used to kill tumorcells directly, block pro-tumorigenic inflammation, and furthermore, canbe used to block angiogenesis. The PD-L1 or PD-L2 component (the firstdomain) in this case would localize the TRAIL to PD-1-positive cells(for example, on antigen-presenting cells or activated T cellsthemselves).

Soluble FasL has also been associated with the induction of apoptosis incertain kinds of tumor cells. Moreover, for certain tumor types,inflammation may actually be pro-tumorigenic. Hence, a FasL fusionprotein can be used to kill tumor cells directly, block pro-tumorigenicinflammation, and furthermore, can be used to block angiogenesis. ThePD-L1 or PD-L2 component (the first domain) in this case would localizethe FasL to PD-1-positive cells (for example, on antigen-presentingcells or activated T cells themselves).

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth.

The fusion proteins according to the invention may be administered toindividuals (such as mammals, including animals and humans) afflictedwith a cellular proliferative disorder such as cancer, and malignant andbenign tumors. In a particular embodiment of the invention, theindividual treated is a human.

Exemplary cancers that may be treated by the fusion proteins,compositions and methods of the invention may also include, but are notlimited to, the following:

cardiac cancers, including, for example sarcoma, e.g., angiosarcoma,fibrosarcoma, rhabdomyosarcoma, and liposarcoma; myxoma; rhabdomyoma;fibroma; lipoma and teratoma;

lung cancers, including, for example, bronchogenic carcinoma, e.g.,squamous cell, undifferentiated small cell, undifferentiated large cell,and adenocarcinoma; alveolar and bronchiolar carcinoma; bronchialadenoma; sarcoma; lymphoma; chondromatous hamartoma; and mesothelioma;

gastrointestinal cancer, including, for example, cancers of theesophagus, e.g., squamous cell carcinoma, adenocarcinoma,leiomyosarcoma, and lymphoma; cancers of the stomach, e.g., carcinoma,lymphoma, and leiomyosarcoma; cancers of the pancreas, e.g., ductaladenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors,and vipoma; cancers of the small bowel, e.g., adenocarcinoma, lymphoma,carcinoid tumors, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma,neurofibroma, and fibroma; cancers of the large bowel, e.g.,adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, andleiomyoma;

genitourinary tract cancers, including, for example, cancers of thekidney, e.g., adenocarcinoma, Wilm's tumor (nephroblastoma), lymphoma,and leukemia; cancers of the bladder and urethra, e.g., squamous cellcarcinoma, transitional cell carcinoma, and adenocarcinoma; cancers ofthe prostate, e.g., adenocarcinoma, and sarcoma; cancer of the testis,e.g., seminoma, teratoma, embryonal carcinoma, teratocarcinoma,choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma,fibroadenoma, adenomatoid tumors, and lipoma;

liver cancers, including, for example, hepatoma, e.g., hepatocellularcarcinoma; cholangiocarcinoma; hepatoblastoma; angiosarcoma;hepatocellular adenoma; and hemangioma;

bone cancers, including, for example, osteogenic sarcoma (osteosarcoma),fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing'ssarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma,malignant giant cell tumor chordoma, osteochrondroma (osteocartilaginousexostoses), benign chondroma, chondroblastoma, chondromyxofibroma,osteoid osteoma and giant cell tumors;

nervous system cancers, including, for example, cancers of the skull,e.g., osteoma, hemangioma, granuloma, xanthoma, and osteitis deformans;cancers of the meninges, e.g., meningioma, meningiosarcoma, andgliomatosis; cancers of the brain, e.g., astrocytoma, medulloblastoma,glioma, ependymoma, germinoma (pinealoma), glioblastoma multiform,oligodendroglioma, schwannoma, retinoblastoma, and congenital tumors;and cancers of the spinal cord, e.g., neurofibroma, meningioma, glioma,and sarcoma;

gynecological cancers, including, for example, cancers of the uterus,e.g., endometrial carcinoma; cancers of the cervix, e.g., cervicalcarcinoma, and pre-tumor cervical dysplasia; cancers of the ovaries,e.g., ovarian carcinoma, including serous cystadenocarcinoma, mucinouscystadenocarcinoma, unclassified carcinoma, granulosa-thecal celltumors, Sertoli-Leydig cell tumors, dysgerminoma, and malignantteratoma; cancers of the vulva, e.g., squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, and melanoma;cancers of the vagina, e.g., clear cell carcinoma, squamous cellcarcinoma, botryoid sarcoma, and embryonal rhabdomyosarcoma; and cancersof the fallopian tubes, e.g., carcinoma;

hematologic cancers, including, for example, cancers of the blood, e.g.,acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblasticleukemia, chronic lymphocytic leukemia, myeloproliferative diseases,multiple myeloma, and myelodysplastic syndrome, Hodgkin's lymphoma,non-Hodgkin's lymphoma (malignant lymphoma) and Waldenström'smacroglobulinemia, angioimmunoblastic T-cell lymphoma (AITL), chroniclymphocytic leukemia (CLL), acute nonlymphocytic leukemia, chroniclymphocytic leukemia, acute granulocytic leukemia, chronic granulocyticleukemia, monocytic leukemia, myeloblastic leukemia, myelocyticleukemia, myeloid granulocytic leukemia, myelomonocytic leukemia,Naegeli leukemia, plasma cell leukemia, plasmacytic leukemia,promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stemcell leukemia, subleukemic leukemia, acute promyelocytic leukemia, adultT-cell leukemia, aleukemic leukemia, aleukocythemic leukemia, basophilicleukemia, blast cell leukemia, bovine leukemia, chronic myelocyticleukemia, leukemia cutis, embryonal leukemia, undifferentiated cellleukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia,hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia,stem cell leukemia, acute monocytic leukemia, leukopenic leukemia,lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia,lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia,mast cell leukemia, megakaryocytic leukemia and micromyeloblasticleukemia;

skin cancers, including, for example, malignant melanoma, basal cellcarcinoma, squamous cell carcinoma, Kaposi's sarcoma, moles dysplasticnevi, lipoma, angioma, dermatofibroma, keloids, psoriasis; and

adrenal gland cancers, including, for example, neuroblastoma.

Cancers may be solid tumors that may or may not be metastatic. Cancersmay also occur, as in leukemia, as a diffuse tissue. Thus, the term“tumor cell”, as provided herein, includes a cell afflicted by any oneof the above identified disorders.

More particular examples of such cancers include kidney or renal cancer,breast cancer, colon cancer, rectal cancer, colorectal cancer, lungcancer including small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung and squamous carcinoma of the lung, squamouscell cancer (e.g. epithelial squamous cell cancer), cervical cancer,ovarian cancer, prostate cancer, liver cancer, bladder cancer, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancerincluding gastrointestinal cancer, gastrointestinal stromal tumors(GIST), pancreatic cancer, head and neck cancer, glioblastoma,retinoblastoma, astrocytoma, thecomas, arrhenoblastomas, hepatoma,hematologic malignancies including non-Hodgkins lymphoma (NHL), multiplemyeloma and acute hematologic malignancies, endometrial or uterinecarcinoma, endometriosis, fibrosarcomas, choriocarcinoma, salivary glandcarcinoma, vulval cancer, thyroid cancer, esophageal carcinomas, hepaticcarcinoma, anal carcinoma, penile carcinoma, nasopharyngeal carcinoma,laryngeal carcinomas, Kaposi's sarcoma, melanoma, skin carcinomas,Schwannoma, oligodendroglioma, neuroblastomas, rhabdomyosarcoma,osteogenic sarcoma, leiomyosarcomas, urinary tract carcinomas, thyroidcarcinomas, Wilm's tumor, as well as B-cell lymphoma (including lowgrade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL)NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL;high grade immunoblastic NHL; high grade lymphoblastic NHL; high gradesmall non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; and post-transplantlymphoproliferative disorder (PTLD), as well as abnormal vascularproliferation associated with phakomatoses, edema (such as thatassociated with brain tumors), and Meigs' syndrome. “Tumor”, as usedherein, refers to all neoplastic cell growth and proliferation, whethermalignant or benign, and all pre-cancerous and cancerous cells andtissues.

Various cell types have been found to express PD-1 and may be targetedby the fusion proteins of the invention. These include reactive T cellsfrom Non-Hodgkins lymphoma, neoplastic B cells from small lymphocyticlymphoma, grade III follicular lymphoma and diffuse large cell lymphomaand immune cells from chronic lymphocytic leukemia (B-CLL) (Xerri L.,Chetaille B, Serriari N, Attias C, Guillaume Y, Arnoulet C and Olive D,Human Pathol, 2008, 39(7):1050-1058).

For treating cancer, a fusion protein of the invention may beadministered to achieve reduction in the number of cancer cells orabsence of the cancer cells; reduction in the tumor size; inhibition(i.e., slow to some extent and preferably stop) of tumor metastasis;inhibition, to some extent, of tumor growth; increase in length ofremission, and/or relief to some extent, one or more of the symptomsassociated with the specific cancer; reduced morbidity and mortality,and improvement in quality of life issues. Treatment may result in acomplete response, defined as disappearance of all signs of cancer, or apartial response, wherein the size of the tumor is decreased, preferablyby more than 50 percent, more preferably by 75%. Treatment may alsoresult in the patient experiencing disease stabilization. Parameters forassessing successful treatment and improvement in the disease arereadily measurable by routine procedures familiar to a physician ofappropriate skill in the art.

The fusion proteins of the present invention are administered in amountseffective to provide improvement in any of the above parameters used tomeasure success in treatment of cancer, and can be readily determined byone skilled in the art. For example, an effective amount is that amountwhich is effective in inducing apoptosis in some cancer cells, or amajority of cancer cells, or substantially all of the patient's cancercells. Other examples of an effective amount include amounts which areeffective in reducing proliferation of tumor cells, of halting tumorprogression via invasion of other tissues, reducing angiogenesis, andreducing inflammation.

In the context of treatment for cancer, the fusion proteins of thepresent invention can optionally be administered to a patient incombination with other chemotherapeutic agents. Suitablechemotherapeutic agents include, for example, alkylating agents such asthiotepa and cyclosphosphamide (CYTOXAN™); alkyl sulfonates such asbusulfan, improsiilfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, trietylenephosphoramide,triethylenethiophosphaoramide and trimethylolomelamine; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, ranimustine;antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine,bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin,carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,5-FU; androgens such as calusterone, dromostanolone propionate,epitiostanol, mepitiostane, testolactone; anti-adrenals such asaminoglutethimide, mitotane, trilostane; folic acid replenisher such asfrolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine;demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes, e.g.paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) anddocetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); Ifosfamide; mitomycin C; mitoxantrone;vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylomithine (DMFO); retinoic acid; esperamicins;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

Chemotherapeutic agents also include anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogensincluding for example tamoxifen, raloxifene, aromatase inhibiting4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,onapristone, and toremifene (Fareston); and anti-androgens such asflutamide, nilutamide, bicalutamide, leuprolide, and goserelin; andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

Chemotherapeutic agents also include chemotherapeutic agents that areable to sensitize tumour cells to TRAIL and overcome TRAIL resistance,such as proteasome inhibitors and histone deacetylase (HDAC) inhibitors,cycloheximide, imatinib mesylate and other protein tyrosine kinaseinhibitors, 17-allylamino-17-demethoxygeldanamycin, arsenic trioxide andX-linked Inhibitors of Apoptosis Protein small molecule antagonists; andpharmaceutically acceptable salts, acids or derivatives of any of these.

Additional information on the methods of cancer treatment is provided inU.S. Pat. No. 7,285,522, incorporated by reference in its entirety.

Accordingly, in a preferred embodiment, the fusion proteins of thepresent invention can be used to treat breast cancer. In anotherpreferred embodiment, the fusion proteins of the invention can be usedto treat colon cancer. In another embodiment, the fusion proteins of theinvention can be used to treat liver cancer. In another preferredembodiment, the fusion proteins of the invention can be used to treatovarian cancer. In another embodiment, the fusion proteins of theinvention can be used to treat leukemia. In another embodiment, thefusion proteins of the invention can be used to treat melanoma.

In further embodiments, the fusion proteins of the present invention canbe used to treat alloimmune diseases, for example graft rejection, orgraft-versus-host or host-versus-graft disease.

The practice of the invention is illustrated by the followingnon-limiting example. The invention should not be construed to belimited solely to the compositions and methods described herein, butshould be construed to include other compositions and methods as well.One of skill in the art will know that other compositions and methodsare available to perform the procedures described herein.

The example below is described with respect to a representativePD-L1-TRAIL fusion protein. However, a person of skill in the art wouldunderstand how to conduct the corresponding experiments withPD-L2-TRAIL, PD-L1-FasL or PD-L2-FasL, or with any other fusion proteinof the invention.

Examples Materials and Methods

Mice 4-6 week old C57BL/6 female mice were purchased from the JacksonLaboratory (Bar Harbor, Me.), and were maintained under pathogen-freeconditions.

Reagents

Plasmids pT2/BH and pNEB 193 UbC-SB 11 are provided by Dr. Perry Hacket(University of Minnesota, Minneapolis), and murine TRAIL cDNAs areobtained from Dr. Hideo Yagita (Juntendo University School of Medicine,Tokyo, Japan). The plasmid pMFneo is obtained from Dr. Herman Waldmann(University of Oxford, Oxford, UK). Mouse MOG 38-50 peptide(GWYRSPFSRVVHL (SEQ ID NO:28)) is synthesized using F-moc solid phasemethods and purified by HPLC at Invitrogen Life Technologies (Carlsbad,Calif.). Pertussis toxin may be purchased from EMD Biosciences (SanDiego, Calif.). The following reagents are purchased from BD Pharmingen(San Diego, Calif.): ELISA Ab pairs for mouse IL-2, IL-4, IL-6,IFN-.gamma. and recombinant mouse IL-2, IL-4, IL-6, IFN-.gamma. An IL-17ELISA Ab pairs is obtained from Southern Biotech (Alabama, USA), andrecombinant mouse IL-17 is purchased from Biosource (Camarillo, Calif.).PE-anti-mouse TRAIL and PE-anti-mouse PD-L1 are purchased fromeBioscience (San Diego, Calif.). Recombinant TRAIL (Super Killer TRAIL™)is purchased from Axxora Platform (San Diego, Calif.).

Plasmid Construction

Chimeric PD-L1-TRAIL and PD-L1-IgG1(mut) coding cassettes areconstructed by PCR, using partially overlapping syntheticoligonucleotides. cDNA encoding amino acids (aa) 19-239 of murine PD-L1(Swiss-prot accession number Q9EP73.1) is joined to cDNA encoding eitheraa 118-291 of murine TRAIL (Swiss-prot accession number P50592) or amutated human IgG1 Fc (Feyl) segment, respectively. For the latter, acDNA encoding human Fcγ1 (Brunschwig E B, Levine E, Trefzer U,Tykocinski M L: Glycosylphosphatidylinositol-modified murine B7-1 andB7-2 retain costimulator function, J Immunol 1995, 155:5498-5505) ismodified by PCR-based site-directed mutagenesis, using oligonucleotidesconfigured to mutate C220→S, C226→S, C229→S, N297→A, E233-→P, L234→V,and L235→A. To express soluble TRAIL, cDNA encoding aa 118-291 of murineTRAIL is used. All of these cDNA segments are subcloned into a pMFneoeukaryotic expression vector downstream of an EF1α promoter region.Coding sequence for luciferase is mobilized with Hind3- and BamH1 frompTAL-Luc (BD Biosciences; San Jose, Calif.), and subcloned into therespective sites of pMFneo.

To generate a derivative Sleeping Beauty expression vector incorporatingwithin the same plasmid both transposon and transposase cassettes, atransposase coding sequence flanked upstream by a ubiquitin C promoteris generated by PCR from pNEB 193 UbC-SB 11 (b.p. 432-2958) and thenligated between the Apa1 and Xho1 sites of pT2/BH vector, which containsa transposon cassette. This new expression vector, incorporating bothtransposase and transposon expression cassettes, is designated pSBC21.Next, cDNAs corresponding to PD-L1-TRAIL, soluble PD-L1,PD-L1-IgG1(mut), soluble TRAIL, or luciferase, each linked to the EF1α,promoter, are subcloned from their respective pMFneo expressionconstructs into the transposon cassette of pSBC21, downstream of thetransposase expression module. All subcloned cDNAs are oriented in thesame direction as the transposase.

Cell Culture and Transfection

Human 293 kidney cells and CHO cells are cultured in DMEM and HAM'SF-12, respectively, supplemented with 100 μg/ml penicillin, 100 Hindstreptomycin, 2 and 10% heat-inactivated fetal bovine serum. 293 cellsare transiently transfected with the PD-L1-TRAIL, soluble PD-L1,PD-L1-IgG1(mut) and soluble TRAIL pMFneo expression plasmids, usingLipofectAMINE™ reagent (Invitrogen Life Technologies, Carlsbad, Calif.).Proteins in conditioned media are resolved by SDS-PAGE and detected byWestern blot analysis. Anti-mouse Ab used for detecting PD-L1 and TRAILare purchased from R&D Systems (Minneapolis, Minn.), respectively.

Induction and Disease Evaluation of EAE EAE is induced according to astandard induction protocol. Stromnes I M, Goverman J M: Activeinduction of experimental allergic encephalomyelitis, Nat Protoc 2006,1:1810-1819. Briefly, female C57BL/6 mice are challenged with a total of300 μg of MOG38-50 peptide (divided into two subcutaneous injections,one on each dorsal flank) in 0.1 ml PBS, emulsified in an equal volumeof CFA containing 4 mg/ml Mycobacterium tuberculosis H37RA (Difco,Detroit, Mich.). These mice are simultaneously injected intravenouslywith 100 ng of pertussis toxin in 0.2 ml PBS. A second intravenousinjection of pertussis toxin (100 ng/mouse) was given 48 h later. Miceare examined daily for signs of EAE and scored.

Cytokine and Proliferation Assay

For cytokine assays, splenocytes are cultured at 1.5×10⁶ cells per wellin 0.2 ml of DMEM with 10% FBS, in the presence or absence of differentconcentrations of MOG38-50 peptide, or 1 μg/ml Con A (Sigma-Aldrich, St.Louis, Mo.). Conditioned media are collected 40 h later, and cytokineconcentrations are determined by quantitative ELISA, using paired mAbspecific for the corresponding cytokines, per the manufacturer'srecommendations (BD Pharmingen, (San Diego, Calif.). Proliferationassays are performed using 0.5×10⁶ cells per well in 96-well plates.[³H] thymidine is added to the cultures at 48 h, and cells are harvested16 h later. Radioactivity is determined using a flatbed β-counter(Wallac).

Hydrodynamic Injection

Mice are injected with pSBC21 vector alone or pSBC21-based expressionconstructs incorporating PD-L1-TRAIL, soluble PD-L1, PD-L1-IgG1(mut),soluble TRAIL, or luciferase coding sequences. Expression plasmids aredissolved in saline in a volume (in ml) equivalent to 10% of body weight(in gm). The entire volume for each animal is injected within 5 sec viatail veins, according to a published protocol. Liu F, Song Y, Liu D;Hydrodynamics-based transfection in animals by systemic administrationof plasmid DNA, Gene Ther 1999, 6:1258-1266. Retro-orbital blood samplesare collected using heparinized glass capillaries. After centrifugation,plasma is recovered and kept at −20° C. until ELISA assays areperformed.

Measurement of Recombinant Proteins in Serum

ELISA assays are performed in 96-well microtitration plates ForPD-L1-TRAIL, soluble PD-L1, and PD-L1-IgG1(mut), purifiedanti-human/mouse PD-L1 receptor Ab from eBioscience (San Diego, Calif.)is used as capture Ab; for soluble TRAIL, anti-mouse TRAIL Ab from R&DSystems (Minneapolis, Minn.) is used as capture Ab. Detecting Ab are:biotin-anti-mouse PD-L1 Ab from eBioscience for PD-L1; biotin-anti-mouseTRAIL Ab from eBioscience for PD-L1-TRAIL and soluble TRAIL; anti-humanIgG, Fcγ fragment-specific Ab from Jackson ImmunoResearch Laboratories(West Grove, Pa.) for PD-L1-IgG1(mut).

Capture Ab diluted in coating buffer (0.1 M carbonate, pH 8.2) isdistributed in microtitration plates and incubated at 4° C. overnight.After washing twice with 0.05% Tween-20 in PBS, wells are incubated foran additional 2 h at RT with PBS-3% albumin to block nonspecific bindingsites. After washing twice again, 100 μl of serum samples are added andincubated at 4° C. overnight. After incubation, wells are rinsed fourtimes and incubated for 1 h with biotinylated detection Ab. For theenzymatic reaction, avidin peroxidase and TMB Microwell peroxidasesubstrate (KPL, Gaithersburg, Md.) are applied sequentially.

In Vivo Bioluminescence Imaging

Images are acquired at 5 h, 24 h, 5 days, 22 days, 34 days, 51 days, and1 year after injection of the luciferase expression plasmid. At the timeof imaging, mice are anesthetized with ketamine/xylazine. D-luciferin(Biotium, Hayward, Calif.) is dissolved in saline and delivered viaintraperitoneal injection before imaging. Mice are then placed in animaging chamber in which the temperature was maintained at 33° C.Bioluminescent images are acquired using the Xenogen in vivo ImagingSystem (IVIS; Xenogen Corp, Alameda, Calif.). Imaging parameters arefield of view of 8 or 10 cm, exposure time of 4 minutes, number ofbinning 16, and fl/stop of 1. For display, the luminescent image(pseudocolor) is overlaid on a photographic image, which delineates theanatomic landmarks.

Measurement of Blood-Brain Barrier (BBB) Permeability

BBB permeability is assessed essentially as described (Prasad R, Giri S,Nath N, Singh I, Singh A K:5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside attenuatesexperimental autoimmune encephalomyelitis via modulation ofendothelial-monocyte interaction, J Neurosci Res 2006, 84:614-625), withsome modifications. Briefly, on days 6 and 13 after MOG challenge, 4%Evans blue dye (Sigma-Aldrich, St. Louis, Mo.) is injected into the tailveins of C57BL/6 mice. After 1 h, animals are anesthetized andtranscardially perfused with saline to remove intravascular dye.Following euthanasia, spinal cords, cerebellums/brainstems and brainsare collected. For quantitative measurements, spinal cords arehomogenized in 1 ml PBS. Samples are centrifuged once at 15,800 g for 30min. 600 μl aliquots of the supernatant are then collected and added to600 ul of 100% TCA (Sigma Aldrich St. Louis, Mo.). This solution isincubated overnight, and centrifuged at 15,800 g for 30 min. Evans blueextravasation is quantified spectrophotometrically (excitation 630 nmand emission 680 nm) in the supernatants.

Preparation and Analysis of Infiltrating Cells from Spinal Cords

Single cell suspensions of spinal cords are prepared as describedpreviously. (Hilliard B, Samoilova E B, Liu T S, Rostami A, Chen Y:Experimental autoimmune encephalomyelitis in NF-kappa B-deficient mice:roles of NF-kappa B in the activation and differentiation ofautoreactive T cells, J Immunol 1999, 163:2937-2943.) Briefly, mice aresacrificed and spinal cords are removed, placed in ice-cold RPMI mediumcontaining 27% Percoll, and pressed through a 70-μm Falcon cellstrainer. The resulting cell suspension is brought to a volume of 50 mlwith additional 27% Percoll, mixed, and centrifuged at 300×g for 15 min.The pellet is kept on ice, while the myelin layer and supernatant aretransferred to a new 50-ml tube, homogenized by shaking, and centrifugedagain at 300×g for 15 min. The cell pellets are then combined and washedthree times in RPMI medium at 4° C. For flow cytometric analysis, singlecell suspensions of recovered cells are incubated for 45 min with thefollowing Ab: FITC-anti-mouse-IFNγ, PE-anti-mouse IL-10, APC-anti-mouseIL-17, APC-Alexa flour 750-anti-mouse CD4, Percp-cy5.5-anti-mouse CD8,and PE-cy7-anti-mouse CD69, all purchased from eBioscience.

Molecular Modeling of the Chimeric PD-L1-TRAIL Protein

A three-dimensional model of the PD-L1-TRAIL protein is generated usingthe crystal structure of TRAIL (pdb code: 1DOG) (Hymowitz S G,Christinger H W, Fuh G, Ultsch M, O'Connell M, Kelley R F, Ashkenazi A,de Vos A M: Triggering cell death: the crystal structure of Apo2L/TRAILin a complex with death receptor 5, Mol Cell 1999, 4:563-571) and amodeled PD-L1 molecule. A three-dimensional model of the ligand bindingdomain (LBD) of PD-L1 is generated using MODELLER. (Marti-Renom M A,Stuart A C, Fiser A, Sanchez R, Melo F, Sali A: Comparative proteinstructure modeling of genes and genomes, Arum Rev Biophys Biomol Struct2000, 29:291-325.)

Flow Cytometry and MTT Assays

Immunostaining is performed at 4° C. with specified Ab suspended in PBScontaining 0.5% BSA and 0.05% sodium azide (NaN3). All flow cytometricanalyses are performed on a FACS Calibur apparatus with Cell Questsoftware and dual laser (488 and 633 nm) excitation (BD Biosciences).The MTT assay is performed according to the manufacturer's protocol(ATCC, Manassas, Va.).

Statistical Analysis

The Student's t test or Mann-Whitney U test is used to determine thestatistical significance of differences A p value of <0.05 wasconsidered to be statistically significant.

Production of Functional PD-L1-TRAIL Protein

Recombinant PD-L1-TRAIL, along with related control proteins (solublePD-L1, PD-L1-IgG1(mut), soluble TRAIL), are produced using a pMFneoeukaryotic expression system. The chimeric PD-L1-TRAIL coding sequencelinked the full extracellular domains of the PD-L1 type I and TRAIL typeII membrane proteins, thereby creating a hybrid soluble type Hype IIfusion protein. To generate the PD-L1-IgG1(mut) coding sequence, severalamino acids within the human IgG1 component are mutated (see Materialand Methods) in order to block FcγR binding (and consequent non-specificdepletion of lymphocytes) and to interfere with N-glycosylation (whichis important for in vivo effector function of human IgG1). Isaacs J D,Greenwood J, Waldmann H: Therapy with monoclonal antibodies. II. Thecontribution of Fc gamma receptor binding and the influence of C(H)1 andC(H)3 domains on in vivo effector function, J Immunol 1998,161:3862-3869. The various pMFneo-based expression constructs aretransiently transfected into 293 cells, and expression and secretion ofthe respective proteins is demonstrated by Western blot analysis ofconditioned media.

To validate the identity of expressed PD-L1-TRAIL, its ability to bindto PD-L1's ligand, PD-1, is assessed. To this end, CHO cells aretransiently transfected with a murine PD-1 cDNA expression construct (inthe pcDNA3 vector), and after 48 h, transfectants are incubated at 4° C.with purified PD-L1-TRAIL or soluble TRAIL. Immunofluorescence and flowcytometric analysis of these cells, using anti-mouse PD-1(for PD-1expression validation) and anti-mouse TRAIL as detecting Ab, may be usedto show binding of PD-L1-TRAIL, but not soluble TRAIL, to cell surfacePD-1 on transfectants.

The functionality of the TRAIL component of PD-L1-TRAIL is determined byevaluating its capacity to induce apoptosis in mouse EL4 T cells, whichconstitutively express PD-1 and TRAIL receptor, measuring Annexin Vstaining by flow cytometry. Recombinant TRAIL (Super Killer TRAIL™) isused as a positive control in this experiment.

Development of a Transposon-Based Expression System for Sustained InVivo Expression of PD-L1-TRAIL

To enable sustained in vivo expression of PD-L1-TRAIL (and controlproteins), the transposon-based ‘Sleeping Beauty (SB)’ expression systemis invoked. This system combines the advantages of plasmid-mediated genedelivery together with an ability to integrate into the chromosome andprovide for sustained transgene expression. To optimize the efficiencyof this expression system, a derivative expression vector is generated,designated pSBC21, that combines within a single plasmid both transposon(accommodating the transgene of interest) and transposase expressioncassettes. Since the relative expression level from the two cassettes isimportant, a number of promoter combinations are screened, anddetermined that a combination of UBC promoter (driving the transposase)and EF1α promoter (driving the transposon cassette), arrayed in tandem,affords strong transgene expression.

The functionality of this unique dual-cassette transposon/transposasevector derivative (with a UBC/EF1α promoter combination) is validatedusing a luciferase reporter. A pLuciferase•SBC21 plasmid, at varyingconcentrations, is administered by hydrodynamic injection to C57BL/6mice. Hydrodynamic injection of transposon-based expression constructsprovides for sustained gene expression in mouse hepatocytes in vivo. LiuF, Song Y, Liu D: Hydrodynamics-based transfection in animals bysystemic administration of plasmid DNA, Gene Ther 1999, 6:1258-1266.Bioluminescent images acquired after administration of luciferase'ssubstrate, D-Luciferin, reveal luciferase expression.

This vector is used for expressing PD-L1-TRAIL, specifically askingwhether levels of PD-L1-TRAIL in serum correlate with the dose ofinjected pPD-L1-TRAIL•SBC21 plasmid. C57BL/6 mice (in experimentalgroups of four) are each treated with a single hydrodynamic injection ofpPD-L1-TRAIL•SBC21 plasmid, in escalating doses (5, 10 or 20 μg ofplasmid). Serum levels of PD-L1-TRAIL are measured by ELISA twenty daysafter plasmid administration, and a dose-dependent increase in serumPD-L1-TRAIL levels is observed, starting with the 10 μg plasmid dose.

PD-L1-TRAIL Suppresses MOG-Induced Autoimmune Encephalomyelitis

The therapeutic potential of PD-L1-TRAIL in a murine EAE disease modelis investigated. To this end, a single encephalitogenic dose of MOG38-50peptide is administered to C57BL/6 mice. Two days after peptideinjection, a single dose of pPD-L1-TRAIL•SBC21 plasmid (50 μg/mouse), orone of four control plasmids (pPD-L1•SBC21, pPD-L1-IgG1(mut)•SBC21,pTRAIL•SBC21, and pSBC21) is administered by hydrodynamic injection. ByELISA, comparable serum levels of expressed proteins in animalshydrodynamically-injected with each of the respective plasmids aredetected. Disease progression in the treated mice is monitored by bothphysical examination and histological analysis of recovered spinalcords.

PD-L1-TRAIL's therapeutic benefit may also be measured from an analysisof day of disease onset and disease incidence.

Assessment of PD-L1-TRAIL is Effectiveness Vs. its Component Parts, inCombination

The question of whether the PD-L1-TRAIL fusion protein's therapeuticefficacy can be recapitulated by administering soluble PD-L1 and TRAILproteins simultaneously is evaluated, using the same EAE model. Two daysafter administering a single encephalitogenic challenge of MOG38-50peptide to C57BL/6 mice, single doses of either pPD-L1-TRAIL•SBC21plasmid (25 pig/mouse) or a mixture of pPD-L1•SBC21 and pTRAIL•SBC21plasmids (25 μg each/mouse) are hydrodynamically injected into theanimals. This way, it may be demonstrated that PD-L1-TRAIL hassubstantial therapeutic benefit in preventing EAE induction.

Assessment of PD-L1-TRAIL Effect on Proliferation and Differentiation ofAutoreactive T Cells

PD-L1-TRAIL's effect on the proliferation and differentiation ofmyelin-specific T cells recovered from treated animals is assessed. Tothis end, splenocytes are recovered 43 days after MOG challenge fromboth PD-L1-TRAIL-treated and control mice receiving vector only. Thesesplenocytes are evaluated in vitro for their proliferation and cytokineproduction in response to MOG38-50 peptide.

Assessment of PD-L1-TRAIL Effect on Infiltration of Inflammatory Cellsinto CNS

A key pathologic feature of EAE is infiltration of inflammatory cellsinto the CNS. PD-L1-TRAIL's effect on this infiltrative process may beassessed. To this end, a comparison may be made of the absolute numberof inflammatory cells, along with the percentage of early activated CD4+and CD8+ cells and of IFNγ-, IL-17- and IL-10-expressing cells, in thespinal cords of PD-L1-TRAIL-versus vector-treated EAE mice on days 7 and14 post-MOG challenge.

Production and Purification of the Human PD-L1-TRAIL Protein

An expression cassette that may be used to produce human PD-L1-TRAIL iscomprised of the coding sequence for the human urokinase signal peptidefollowed by the coding sequence for human PD-L1-TRAIL. The codingsequences are codon-optimized for enhanced expression in Chinese HamsterOvary (CHO) cell-lines. The DNA coding sequence is synthesized and thensub-cloned into a mammalian expression vector designed for chromosomalintegration and optimized for high level expression in CHO cells.

The PD-L1-TRAIL expression vector is transfected into CHO—S cells, and aclone pool is isolated for initial expression analysis. Out of thisclone pool, a high-producing clone is isolated, and expression of thePD-L1-TRAIL protein is analyzed by various methods such as ELISA,SDS-PAGE and Western Blots. Production levels are optimized to reachexpression of approximately 100 mg of PD-L1-TRAIL per liter offermentation media.

Western blot analysis is performed for shake flask culture mediumsamples obtained from the PD-L1-TRAIL clone grown in various media. TheWestern blot is probed using a commercial anti-human TRAIL/TNFSF10 Ab asprimary detecting antibody.

A high-yield, multi-step chromatographic purification may be used forthe isolation of highly-purified PD-L1-TRAIL protein. The processincludes an efficient capture step, an anion-exchange chromatographystep, and then a final buffer exchange step, the latter carrying theproduct into the formulation buffer.

A seven-liter production fermentation followed by the above purificationprocess, yields approximately 300 mg of purified PD-L1-TRAIL which maybe used for a series of in-vitro and in-vivo experiments indicatedbelow.

EAE Experiments, with Human PD-L1-Trail-Experimental Procedures

EAE is induced in 8-week-old female C57BL/6 mice by injectingsubcutaneously, into the left para-lumbar region, 125 ug of myelinoligodendrocyte glycoprotein 35-55 (MOG 35-55) peptide (synthesized bySigma Laboratories, Israel), emulsified in complete Freund's adjuvant(CFA) containing 5 mg/ml heat-killed Mycobacterium tuberculosis.Immediately thereafter, and, again, at 48 hours, the mice are inoculatedwith 300 ng of pertussis toxin. An additional injection of MOG 35-55peptide in CFA is delivered 7 days later into the right para-lumbarregion. From day 0 to day 8 mice are injected subcutaneously with 50,100 or 200 micrograms a day of human PD-L1-TRAIL (human PD-L1 and humanTRAIL bind mouse PD-1 and mouse TRAIL receptor, respectively) orvehicle, in two equal doses (n=4 in each group). On day 9 the mice aretreated for the last time, and sacrificed an hour later. Spleens areharvested and weighed. Pooled lymph node cells (LNCs) are prepared frominguinal, axillary and mesenteric lymph nodes or from spleens of micethat have been inoculated 9 days earlier with MOG35-55 peptide in CFAwith or without PD-L1-TRAIL treatment. The ex vivo response of thelymphocytes is assayed in triplicate wells of 96-well flat-bottomplates. A total of 2×10⁵ cells, suspended in 0.2 ml RPMI supplementedwith 1% penicillin streptomycin, 1% glutamine and 5% fetal calf serum(FCS) and beta-mercapto-ethanol are added to each well. After 48 hrs, 1μCi 3(H)Thymidine (Amersham, UK) is added to each well and the platesare incubated for an additional 18 hrs. Plates are then harvested with asemi-automatic harvester onto a glass fiber filter and the radioactivityis determined by liquid scintillation. The results are expressed asStimulation Index (SI) according to the equation: SI=Mean cpm of thestimulated cells/mean cpm of the unstimulated cells.

Pooled spleen lymphocytes are isolated, using a Ficoll-Hipaque gradient,on day 9 from MOG-immunized mice treated with PD-L1-Trail (n=4 animalsin each group). Recovered cells are stained with methylene blue andcounted.

Pooled lymphocytes isolated from lymph nodes recovered on day 9 ofPD-L1-Trail treatment in MOG-immunized mice (n=4 in each group) arestimulated for 72 hrs with MOG peptide. Cultures are pulsed with[³H]-thymidine 18 hrs before the end of incubation. Proliferation isestimated by [³H]thymidine incorporation and is expressed as stimulationindex (mean cpm of stimulated cells/mean cpm of non-stimulated cells;SI>2 represents significant stimulation).

EAE is induced by MOG challenge, as described above. On day 10 after MOGadministration, mice (10 in each group) are treated with either vehicleor PD-L1-Trail at 25, 50 and 100 μg/day, in two divided doses. Mice arefollowed daily for the evaluation of their clinical disease scores. Theclinical status of mice is graded as follows: 0, no signs of disease; 1,tail weakness; 2, hind limb weakness sufficient to impair righting; 3,hind limb paresis; 4, paraplegia with forelimb weakness; 5,quadriplegia; 6, death. Treatment is stopped on day 26 after diseaseinduction (day 16 of PD-L1-Trail treatment).

Collagen-Induced Arthritis with PD-L1-Trail—Experimental Procedures

DBA1 male mice are challenged twice (3 weeks apart) with 200 ug of typeII collagen purified from bovine articular cartilage and emulsified incomplete Freund's adjuvant (CFA: Difco Labs), via intradermal injectionat the base of the tail. Mice are followed daily and monitored forswelling and/or erythema in one or more limbs. On the day of diseaseonset, mice are randomized for control group (treated with vehicle only)and treatment groups (one daily dose of 100 or 200 ug/mice/d ofPD-L1-Trail). Both vehicle and PD-L1-Trail are administeredsubcutaneously. Injections are given daily for 14 days to the controland 100 ug group, and for 7 days to the 200 ug group. Mice are followeddaily for 14 days from disease onset, and then every 3 days. Swelling inall 4 limbs is measured using a microcaliper, and compared to healthy,age-matched mice. The delta of swelling in each limb is calculated, andthese deltas are summed into a score (disease index).

Assay for PD-L1-TRAIL-Driven Cytotoxicity Against Cancer Cells

Tumor cell cytotoxicity mediated by purified untagged human PD-L1-TRAILprotein is studied with several human tumor cell types. Human leukemiacell lines are incubated with increasing concentrations of purifiedPD-L1-TRAIL, and the EC50 is measured.

Assessment of PD-L1-TRAIL's Tumoricidal Activity vs. its Component Partsin Combination

The tumoricidal effect of the PD-L1-TRAIL fusion protein is compared tothe effect of its component parts added in combination. To this end,cell viability of the hepatoma cancer cell line SK-Hep1 is measuredfollowing incubation with either purified PD-L1-TRAIL, solubleextracellular domain of TRAIL (sTRAIL) alone, soluble PD-L1 fused to theFc domain of IgG1 (PD-L1-Fc), or the combination of both(PD-L1-Fc+sTRAIL), at similar molar concentrations.

Apoptosis Assay

Cells of the human hepatoma cancer cell line SK-Hep1 are incubated withincreasing concentrations of purified PD-L1-TRAIL, soluble PD-L1 fusedto the Fc domain of IgG (PD-L1-Fc) alone, the soluble extracellulardomain of TRAIL (sTRAIL) alone, or a combination of both(PD-L1-Fc+sTRAIL). Following incubation with the respective proteins,the treated cells are analyzed by FACS to determine the percentage ofcells undergoing apoptosis, as assessed by annexin V/PI staining.

The disclosures of each and every patent, patent application,publication, GenBank UniProtKB, or SwissProt record cited herein arehereby incorporated herein by reference in their entirety.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. While theinvention has been disclosed with reference to specific embodiments, itis apparent that other embodiments and variations of this invention maybe devised by others skilled in the art without departing from the truespirit and scope used in the practice of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

What is claimed is:
 1. A fusion protein comprising a first domain and asecond domain, wherein the first domain comprises a polypeptide thatbinds to and triggers PD-1 and the second domain comprises a polypeptidethat binds to and triggers a TRAIL receptor or Fas.
 2. The fusionprotein according to claim 1, wherein the polypeptide that binds to andtriggers PD-1 comprises at least a portion of the extracellular domainof PD-L1 or PD-L2 and the second domain comprises at least a portion ofthe extracellular domain of TRAIL or Fas ligand.
 3. The fusion proteinaccording to claim 1, wherein the fusion protein comprises at least aportion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11 or SEQ ID NO:12.
 4. The fusion protein according to claim 1comprising a first domain and a second domain, wherein the first domaincomprises a portion of the extracellular domain of PD-L1 or PD-L2comprising at least 20 contiguous amino acids of said extracellulardomain, and the second domain comprises a portion of the extracellulardomain of TRAIL or Fas ligand comprising at least 20 contiguous aminoacids of said extracellular domain.
 5. The fusion protein according toclaim 1, wherein the first domain comprises human PD-L1 or human PD-L2,or a fragment thereof capable of binding to and triggering PD-1, and thesecond domain comprises human TRAIL or human Fas ligand, or a fragmentthereof.
 6. The fusion protein according to claim 1, wherein the firstdomain and the second domain are connected via a linker.
 7. The fusionprotein according to claim 6, wherein the linker is a protein linker. 8.The fusion protein according to according to claim 1, further comprisinga trimerization domain.
 9. A pharmaceutical composition comprising apharmaceutically acceptable carrier and a fusion protein according toclaim
 1. 10. A method of treating an autoimmune or alloimmune diseasecomprising administering a therapeutically effective amount of a fusionprotein according to claim 1 to a patient in need of such treatment. 11.The method of claim 10, wherein the autoimmune disease is multiplesclerosis.
 12. A method of treating an inflammatory disease comprisingadministering a therapeutically effective amount of a fusion proteinaccording to claim 1 to a patient in need of such treatment.
 13. Amethod of inhibiting proliferation and differentiation of T cells, Bcells, mast cells, antigen presenting cells, dendritic cells or NK cellsin a patient in need thereof, the method comprising the step ofadministering to said patient a therapeutically effective amount of afusion protein according to claim
 1. 14. A method of treating cancercomprising administering to a patient in need of such treatment atherapeutically effective amount of a fusion protein according toclaim
 1. 15. A method of treating autoimmune disease or alloimmunedisease comprising administering to a patient in need of such treatmentan effective amount of a genetic sequence encoding a fusion proteinaccording to claim
 1. 16. A method of treating inflammatory diseasecomprising administering to a patient in need of such treatment aneffective amount of a genetic sequence encoding a fusion proteinaccording to claim
 1. 17. A method of treating cancer comprisingadministering to a patient in need of such treatment an effective amountof a genetic sequence encoding the fusion protein according to claim 1.18. The method of claim 10, wherein administration of said fusionprotein is parenteral.
 19. The method of claim 12, whereinadministration of said fusion protein is parenteral.
 20. The method ofclaim 13, wherein administration of said fusion protein is parenteral.21. The method of claim 14, wherein administration of said fusionprotein is parenteral.
 22. (canceled)
 23. (canceled)
 24. (canceled) 25.(canceled)
 26. (canceled)
 27. (canceled)